Capture of nucleic acids using a nucleic acid-guided nuclease-based system

ABSTRACT

Provided herein are methods and compositions for the capture of nucleic acids, for example by using a nucleic acid-guided nuclease-based system.

CROSS REFERENCE TO RELATED APPLICATIONS

This is a division of U.S. application Ser. No. 15/520,052, filed onApr. 18, 2017 (now allowed), which is a U.S. National Stage Applicationunder 35 U.S.C. § 371 of International Application No.PCT/US2016/047631, filed on Aug. 18, 2016, which claims priority to U.S.Provisional Application Ser. No. 62/207,359, filed on Aug. 19, 2015,each of which is incorporated herein by reference in its entirety.

INCORPORATION OF THE SEQUENCE LISTING

The contents of the text file named “ARCB_00201US_SeqList.txt”, whichwas created on Nov. 27, 2019 and is 6 KB in size, are herebyincorporated by reference in their entirety.

BACKGROUND OF THE INVENTION

Targeted sequencing of specific regions of the genome continues to be ofinterest to researchers, particularly in a clinical setting. Clinicaldiagnosis of genetic disease, cancer, and many research projects rely ontargeted sequencing to enable high coverage sequencing of targeted siteswhile reducing sequencing costs. Currently, the primary methods used forthis purpose are 1) hybridization-based enrichment, and 2) multiplexPCR. In the former approach, biotin-labeled short oligonucleotide probesare used to “pull out” sequences of interest from a library. Thisprocess can be time-consuming, expensive, and require many hands-onsteps. Furthermore, often some “off-target” sequences can remain in theresulting product. The multiplex PCR-based approach can be faster, butthe number of targets can be limited and the cost can be high. Neededare methods for the efficient capture of nucleic acid regions ofinterest that are easy, specific, rapid, and inexpensive. Providedherein are methods and compositions that address this need.

All patents, patent applications, publications, documents, web links,and articles cited herein are incorporated herein by reference in theirentireties.

BRIEF SUMMARY OF THE INVENTION

Provided herein are methods and compositions that allow for theselective capture of nucleic acid sequences of interest. The nucleicacids may contain DNA or RNA. The methods and compositions providedherein are particularly useful for working with complex nucleic acidsamples.

In one aspect, the invention provides a method of capturing targetnucleic acid sequences comprising: (a) providing a sample comprising aplurality of adapter-ligated nucleic acids, wherein the nucleic acidsare ligated to a first adapter at one end and ligated to a secondadapter at the other end; (b) contacting the sample with a plurality ofnucleic acid-guided nuclease-gNA complexes, wherein the gNAs arecomplementary to targeted sites of interest contained in a subset of thenucleic acids, thereby generating a plurality of nucleic acid fragmentsligated to a first or second adapter at one end and no adapter at theother end; and (c) contacting the plurality of nucleic acid fragmentswith third adapters, thereby generating a plurality of nucleic acidfragments ligated to either the first or second adapter at one end andthe third adapter at the other end. In one embodiment, the nucleicacid-guided nuclease is a CRISPR/Cas system protein. In one embodiment,the nucleic acid-guided nuclease is a non-CRISPR/Cas system protein. Inone embodiment, the nucleic acid-guided nuclease is selected from thegroup consisting of CAS Class I Type I, CAS Class I Type III, CAS ClassI Type IV, CAS Class II Type II, and CAS Class II Type V. In oneembodiment, the nucleic acid-guided nuclease is selected from the groupconsisting of Cas9, Cpf1, Cas3, Cas8a-c, Cas10, Cse1, Csy1, Csn2, Cas4,Csm2, Cm5, Csf1, C2c2, and NgAgo. In one embodiment, the gNAs are gRNAs.In one embodiment, the gNAs are gDNAs. In one embodiment, the contactingwith a plurality of nucleic acid-guided nuclease-gNA complexes cleavesthe targeted sites of interest contained in a subset of the nucleicacids, thereby generating a plurality of nucleic acid fragmentscomprising a first or second adapter at one end and no adapter at theother end. In one embodiment the method further comprises amplifying theproduct of step (c) using first or second and third adapter-specificPCR. In one embodiment the nucleic acids are selected from the groupconsisting of single stranded DNA, double stranded DNA, single strandedRNA, double stranded RNA, and a DNA/RNA hybrid. In one embodiment thenucleic acids are double stranded DNA. In one embodiment the nucleicacids are from genomic DNA. In one embodiment the genomic DNA is human.In one embodiment the nucleic acids adapter-ligated ends are from 20 bpto 5000 bp in length. In one embodiment the targeted sites of interestare single nucleotide polymorphisms (SNPs), short tandem repeats (STRs),cancer genes, inserts, deletions, structural variations, exons, geneticmutations, or regulatory regions. In one embodiment amplified productsare used for cloning, sequencing, or genotyping. In one embodiment theadapters are from 20 bp to 100 bp in length. In one embodiment theadapters comprise primer binding sites. In one embodiment the adapterscomprise sequencing adapters or restriction sites. In one embodiment thetargeted sites of interest represent less than 50% of the total nucleicacid in the sample. In one embodiment the sample is obtained from abiological sample a clinical sample, a forensic sample, or anenvironmental sample. In one embodiment the first and second adaptersare identical. In one embodiment the first and second adapters aredifferent. In one embodiment the sample comprises a sequencing library.

In another aspect, the invention provides a method of introducinglabeled nucleotides at targeted sites of interest comprising: (a)providing a sample comprising a plurality of nucleic acid fragments; (b)contacting the sample with a plurality of nucleic acid-guided nucleasenickase-gNA complexes wherein the gNAs are complementary to targetedsites of interest in the nucleic acid fragments, thereby generating aplurality of nicked nucleic acid fragments at the targeted sites ofinterest; and (c) contacting the plurality of nicked nucleic acidfragments with an enzyme capable of initiating nucleic acid synthesis ata nicked site, and labeled nucleotides, thereby generating a pluralityof nucleic acid fragments comprising labeled nucleotides in the targetedsites of interest. In one embodiment, the nucleic acid-guided nucleasenickase is selected from the group consisting of CAS Class I Type Inickase, CAS Class I Type III nickase, CAS Class I Type IV nickase, CASClass II Type II nickase, and CAS Class II Type V nickase. In oneembodiment, the nucleic acid-guided nuclease nickase is selected fromthe group consisting of Cas9 nickase, Cpf1 nickase, Cas3 nickase,Cas8a-c nickase, Cas10 nickase, Cse1 nickase, Csy1 nickase, Csn2nickase, Cas4 nickase, Csm2 nickase, Cm5 nickase, Csf1 nickase, C2C2nickase, and NgAgo nickase. In one embodiment, the the gNAs are gRNAs.In one embodiment, the gNAs are gDNAs. In one embodiment the nucleicacid fragments are selected from the group consisting of single strandedDNA fragments, double stranded DNA fragments, single stranded RNAfragments, double stranded RNA fragments, and a DNA/RNA hybridfragments. In one embodiment the nucleic fragments are double strandedDNA fragments. In one embodiment double stranded DNA fragments are fromgenomic DNA. In one embodiment the genomic DNA is human. In oneembodiment the nucleic acid fragments are from 20 bp to 5000 bp inlength. In one embodiment the targeted sites of interest are singlenucleotide polymorphisms (SNPs), short tandem repeats (STRs), cancergenes, inserts, deletions, structural variations, exons, geneticmutations, or regulatory regions. In one embodiment the targeted sitesof interest represent less than 50% of the total nucleic acid in thesample. In one embodiment the sample is obtained from a biologicalsample, a clinical sample, a forensic sample, or an environmentalsample. In one embodiment the labeled nucleotides are biotinylatednucleotides. In one embodiment, the labeled nucleotides are part of anantibody conjugate pair. In one embodiment the method further comprisescontacting the nucleic acid fragments comprising biotinylatednucleotides with avidin or strepavidin, thereby capturing the targetednucleic acid sites of interest. In one embodiment the enzyme capable ofinitiating nucleic acid synthesis at a nicked site is DNA Polymerase I,a Klenow fragment, a TAQ polymerase, or a Bst DNA Polymerase. In oneembodiment the Cas9 nickase nicks the 5′ end of the nucleic acidfragments. In one embodiment the nucleic acid fragments are from 20 bpto 5000 bp in length. In one embodiment the sample is obtained from abiological sample a clinical sample, a forensic sample, or anenvironmental sample.

In another aspect, the invention provides a method of capturing targetnucleic acid sequences of interest comprising: (a) providing a samplecomprising a plurality of adapter-ligated nucleic acids, wherein thenucleic acids are ligated to a first adapter at one end and are ligatedto a second adapter at the other end; and (b) contacting the sample witha plurality of catalytically dead nucleic acid-guided nuclease-gNAcomplexes, wherein the catalytically dead nucleic acid-guided nucleaseis fused to a transposase, wherein the gRNAs are complementary totargeted sites of interest contained in a subset of the nucleic acids,and wherein the complexes are loaded with a plurality of third adapters,to generate a plurality of nucleic acids fragments comprising either afirst or second adapter at one end, and a third adapter at the otherend. In one embodiment the method further comprises amplifying theproduct of step (b) using first or second adapter and thirdadapter-specific PCR. In one embodiment, the catalytically dead nucleicacid-guided nuclease is derived from a CRISPR/Cas system protein. In oneembodiment, the catalytically dead nucleic acid-guided nuclease isderived from a non-CRISPR/Cas system protein. In one embodiment, thecatalytically-dead nucleic acid-guided nuclease is selected from thegroup consisting of dead CAS Class I Type I, dead CAS Class I Type III,dead CAS Class I Type IV, dead CAS Class II Type II, and dead CAS ClassII Type V. In one embodiment, the catalytically dead nucleic acid-guidednuclease is selected from the group consisting of dCas9, dCpf1, dCas3,dCas8a-c, dCas10, dCse1, dCsy1, dCsn2, dCas4, dCsm2, dCm5, dCsf1, dC2C2,and dNgAgo. In one embodiment, the gNAs are gRNAs. In one embodiment,the gNAs are gDNAs. In one embodiment the nucleic acids sequences arefrom genomic DNA. In one embodiment the genomic DNA is human. In oneembodiment the catalytically dead nucleic acid-guided nuclease is fusedto the N-terminus of the transposase. In one embodiment thecatalytically dead nucleic acid-guided nuclease is fused to theC-terminus of the transposase. In one embodiment the adapter-ligatednucleic acids are 20 bp-5000 bp. In one embodiment the contacting ofstep (b) allows for the insertion of the second adapter into thetargeted nucleic acid sequences. In one embodiment the targeted sites ofinterest are single nucleotide polymorphisms (SNPs), short tandemrepeats (STRs), cancer genes, inserts, deletions, structural variations,exons, genetic mutations, or regulatory regions. In one embodiment theamplified products are used for cloning, sequencing, or genotyping. Inone embodiment the adapters are from 20 bp to 100 bp in length. In oneembodiment the adapters comprise primer binding sites. In one embodimentthe adapters comprise sequencing adapters or restriction sites. In oneembodiment the targeted sites of interest represent less than 50% of thetotal nucleic acid in the sample. In one embodiment the sample isobtained from a biological sample a clinical sample, a forensic sample,or an environmental sample.

In another aspect, the invention provides a method of capturing targetnucleic acid sequences of interest comprising: (a) providing a samplecomprising a plurality of adapter-ligated nucleic acids, wherein thenucleic acids are ligated to the adapter at the 5′ end and 3′ ends; (b)contacting the sample with a plurality of catalytically dead nucleicacid-guided nuclease-gNA complexes, wherein the gNAs are complementaryto targeted sites of interest contained in a subset of the nucleicacids, thereby generating a plurality of nucleic acids adapter-ligatedat the 5′ and 3′ ends, bound to a catalytically dead nucleic acid-guidednuclease-gNA complex; and (c) contacting the sample with a plurality ofcatalytically dead nucleic acid-guided nuclease-gNA complexes, whereinthe gNAs are complementary to both targeted sites of interest andtargeted sites not of interest in the nucleic acids, thereby generatinga plurality of nucleic acid fragments comprising nucleic acid sequencesnot of interest, adapter ligated at only one of the 5′ or 3′ ends. Inone embodiment the contacting of step (c) does not displace theplurality of nucleic acids adapter-ligated at the 5′ and 3′ ends, boundto a dCAS9-gRNA complex of step (b). In one embodiment the contacting instep (d) cleaves the targeted sites not of interest contained in asubset of the nucleic acids, thereby generating a plurality of nucleicacid fragments comprising nucleic acid sequences not of interest,adapter ligated at only one of the 5′ or 3′ ends. In one embodiment themethod further comprises removing the bound dCAS9-gRNA complex andamplifying the product of step (b) using adapter-specific PCR. In oneembodiment the catalytically dead nucleic acid-guided nuclease is aCRISPR/Cas system protein. In one embodiment the catalytically deadnucleic acid-guided nuclease is a non-CRISPR/Cas system protein. In oneembodiment, the catalytically-dead nucleic acid-guided nuclease isselected from the group consisting of dead CAS Class I Type I, dead CASClass I Type III, dead CAS Class I Type IV, dead CAS Class II Type II,and dead CAS Class II Type V. In one embodiment the catalytically deadnucleic acid-guided nuclease is selected from the group consisting ofdCas9, dCpf1, dCas3, dCas8a-c, dCas10, dCse1, dCsy1, dCsn2, dCas4,dCsm2, dCm5, dCsf1, dC2C2, and dNgAgo. In one embodiment the gNAs aregRNAs. In one embodiment the gNAs are gDNAs. In one embodiment thenucleic acids are selected from the group consisting of single strandedDNA, double stranded DNA, single stranded RNA, double stranded RNA, anda DNA/RNA hybrid. In one embodiment the nucleic acids are doublestranded DNA. In one embodiment the nucleic acids are from genomic DNA.In one embodiment the genomic DNA is human. In one embodiment thenucleic acids adapter-ligated at the 5′ends and the 3′ends are from 20bp to 5000 bp in length. In one embodiment the targeted sites ofinterest are single nucleotide polymorphisms (SNPs), short tandemrepeats (STRs), cancer genes, inserts, deletions, structural variations,exons, genetic mutations, or regulatory regions. In one embodiment theamplified products are used for cloning, sequencing, or genotyping. Inone embodiment the adapters are from 20 bp to 100 bp in length. In oneembodiment the adapters comprise primer binding sites. In one embodimentthe adapters comprise sequencing adapters or restriction sites. In oneembodiment the targeted sites of interest represent less than 50% of thetotal nucleic acid in the sample. In one embodiment the sample isobtained from a biological sample, a clinical sample, a forensic sample,or an environmental sample.

In another aspect, the invention provides a method of capturing targetDNA sequences of interest comprising: (a) providing a sample comprisinga plurality of nucleic acid sequences, wherein the nucleic acidsequences comprise methylated nucleotides, and wherein the nucleic acidsequences are adapter ligated on the 5′ and 3′ ends; (b) contacting thesample with a plurality of nucleic acid-guided nuclease nickase-gNAcomplexes, wherein the gNAs are complementary to targeted sites ofinterest in a subset of the nucleic acid sequences, thereby generating aplurality of nicked sites of interest in the subset of the nucleic acidsequences, and wherein the target nucleic acid sequences are adapterligated on the 5′ and 3′ ends; (c) contacting the sample with an enzymecapable of initiating DNA synthesis at a nicked site, and unmethylatednucleotides, thereby generating a plurality of nucleic acid sequencescomprising unmethylated nucleotides in the targeted sites of interestand wherein the nucleic acid sequences are adapter ligated on the 5′ and3′ ends; and (d) contacting the sample with an enzyme capable of cuttingmethylated nucleic acids, thereby generating a plurality of nucleic acidfragments comprising methylated nucleic acids, wherein the plurality ofnucleic acid fragments comprising methylated nucleic acids that areadapter ligated on at most one of the 5′ and 3′ ends. In one embodiment,the nucleic acid-guided nuclease nickase is selected from the groupconsisting of CAS Class I Type I nickase, CAS Class I Type III nickase,CAS Class I Type IV nickase, CAS Class II Type II nickase, and CAS ClassII Type V nickase. In one embodiment the nucleic acid-guided nucleasenickase is selected from the group consisting of Cas9 nickase, Cpf1nickase, Cas3 nickase, Cas8a-c nickase, Cas10 nickase, Cse1 nickase,Csy1 nickase, Csn2 nickase, Cas4 nickase, Csm2 nickase, Cm5 nickase,Csf1 nickase, C2c2 nickase, and NgAgo nickase. In one embodiment thegNAs are gRNAs. In one embodiment the gNAs are gDNAs. In one embodimentthe DNA is double stranded DNA. In one embodiment the double strandedDNA is from genomic DNA. In one embodiment the genomic DNA is human. Inone embodiment the DNA sequences are from 20 bp to 5000 bp in length. Inone embodiment the targeted sites of interest are single nucleotidepolymorphisms (SNPs), short tandem repeats (STRs), cancer genes,inserts, deletions, structural variations, exons, genetic mutations, orregulatory regions. In one embodiment the targeted sites of interestrepresent less than 50% of the total DNA in the sample. In oneembodiment the sample is obtained from a biological sample, a clinicalsample, a forensic sample, or an environmental sample. In one embodimentthe enzyme capable of initiating nucleic acid synthesis at a nicked siteis DNA Polymerase I, Klenow fragment a TAQ polymerase, or a Bst DNAPolymerase. In one embodiment the nucleic acid-guided nuclease nickasenicks the 5′ end of the DNA sequences. In one embodiment the enzymecapable of cutting methylated DNA is DpnI.

In another aspect, the invention provides a method of capturing targetDNA sequences of interest comprising: (a) contacting the sample with aplurality of nucleic acid-guided nuclease nickase-gNA complexes, whereinthe gNAs are complementary to targeted sites flanking a region ofinterest in a subset of the DNA sequences, thereby generating aplurality of nicked DNA at sites adjacent to the regions of interest (b)heating to 65° C. to cause nicks in close proximity to generate a doublestranded break (c) contacting these double stranded breaks with athermostable ligase thereby allowing ligation of adapter sequences atthese sites only and (d), repeating these three steps to place a secondadapter on the other side of the region of interest, thus allowingenrichment of the region of interest. In one embodiment, the gNAs aregRNAs. In one embodiment, the gNAs are gDNAs. In one embodiment, thenucleic acid-guided nuclease nickase is selected from the groupconsisting of CAS Class I Type I nickase, CAS Class I Type III nickase,CAS Class I Type IV nickase, CAS Class II Type II nickase, and CAS ClassII Type V nickase. In one embodiment the nucleic acid-guided nucleasenickase is selected from the group consisting of Cas9 nickase, Cpf1nickase, Cas3 nickase, Cas8a-c nickase, Cas10 nickase, Cse1 nickase,Csy1 nickase, Csn2 nickase, Cas4 nickase, Csm2 nickase, Cm5 nickase,Csf1 nickase, C2C2 nickase, and NgAgo nickase. In one embodiment the DNAis double stranded DNA. In one embodiment the double stranded DNA isfrom genomic DNA. In one embodiment the genomic DNA is human. In oneembodiment the DNA sequences are from 20 bp to 5000 bp in length. In oneembodiment the targeted sites of interest are single nucleotidepolymorphisms (SNPs), short tandem repeats (STRs), cancer genes,inserts, deletions, structural variations, exons, genetic mutations, orregulatory regions. In one embodiment the targeted sites of interestrepresent less than 50% of the total DNA in the sample. In oneembodiment the sample is obtained from a biological sample, a clinicalsample, a forensic sample, or an environmental sample. In one embodimentthe ligase capable of contacting the double stranded break isThermostable 5′App DNA/RNA ligase, or T4 RNA ligase. In one embodimentthe nucleic acid-guided nuclease nickase nicks the 5′ end of the DNAsequences. In one embodiment the enzyme capable of cutting methylatedDNA is DpnI.

In another aspect, the invention provides a method of enriching a samplefor sequences of interest, comprising: (a) providing a sample comprisingsequences of interest and targeted sequences for depletion, wherein thesequences of interest comprise less than 50% of the sample; and (b)contacting the sample with a plurality of either nucleic acid-guided RNAendonuclease-gRNA complexes or a plurality of nucleic acid-guided DNAendonuclease-gDNA complexes, wherein the gRNAs and gDNAs arecomplementary to the targeted sequences, and whereby the targetedsequences are cleaved. In one embodiment, the method further comprisesextracting the sequences of interest and the targeted sequences fordepletion from the sample. In one embodiment, the method furthercomprises fragmenting the extracted sequences. In one embodiment, thecleaved targeted sequences are removed by size-exclusion. In oneembodiment, the sample is any one of a biological sample, a clinicalsample, a forensic sample or an environmental sample. In one embodiment,the sample comprises host nucleic acid sequences targeted for depletionand non-host nucleic acid sequences of interest. In one embodiment, thenon-host nucleic acid sequences comprise microbial nucleic acidsequences. In one embodiment, the microbial nucleic acid sequences arebacterial, viral or eukaryotic parasitic nucleic acid sequences. In oneembodiment, the gRNAs and gDNAs are complementary to ribosomal RNAsequences, spliced transcripts, unspliced transcripts, introns, exons,or noncoding RNAs. In one embodiment, the extracted nucleic acidsinclude single-stranded or double-stranded RNA. In one embodiment, theextracted nucleic acids include single-stranded or double-stranded DNA.In one embodiment, the sequences of interest comprise less than 10% ofthe extracted nucleic acids. In one embodiment, the nucleic acid-guidedRNA endonuclease comprises C2c2. In one embodiment, the C2c2 iscatalytically dead. In one embodiment, the nucleic acid-guided DNAendonuclease comprises NgAgo. In one embodiment, the NgAgo iscatalytically dead. In one embodiment, the sample is selected from wholeblood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth,bone, fingernails, feces, urine, tissue, and a biopsy.

In another aspect, the invention provides a method of enriching a samplecomprising: (a) providing a sample comprising host nucleic acids andnon-host nucleic acids; (b) contacting the sample with a plurality ofnucleic acid-guided RNA endonuclease-gRNA complexes or a plurality ofnucleic acid-guided DNA endonuclease-gDNA complexes, wherein the gRNAsand gDNAs are complementary to targeted sites in the host nucleic acids,and (c) enriching the sample for non-host nucleic acids. In oneembodiment, the nucleic acid-guided RNA endonuclease comprises C2c2. Inone embodiment, the nucleic acid-guided RNA endonuclease comprisescatalytically dead C2c2. In one embodiment, the nucleic acid-guided DNAendonuclease comprises NgAgo. In one embodiment, the nucleic acid-guidedDNA endonuclease comprises catalytically dead NgAgo. In one embodiment,the host is selected from the group consisting of a human, cow, horse,sheep, pig, monkey, dog, cat, gerbil, bird, mouse, and rat. In oneembodiment, the non-host is a prokaryotic organism. In one embodiment,the non-host is selected from the group consisting of a eukaryote, avirus, a bacterium, a fungus, and a protozoan. In one embodiment, theadapter-ligated host nucleic acids and non-host nucleic acids range from50 bp to 1000 bp in length. In one embodiment, the non-host nucleicacids comprise less than 50% of the total nucleic acids in the sample.In one embodiment, the sample is any one of a biological sample, aclinical sample, a forensic sample or an environmental sample. In oneembodiment, step (c) comprises reverse-transcribing the product of step(b) into cDNA. In one embodiment, step (c) comprises removing the hostnucleic acids by size-exclusion. In one embodiment, step (c) comprisesremoving the host nucleic acids with the use of biotin. In oneembodiment, the sample is selected from whole blood, plasma, serum,tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails,feces, urine, tissue, and a biopsy.

In another aspect, the invention provides a method of using a nucleicacid-guided RNA endonuclease to enrich for a target in an RNA sampleusing labeled, catalytically dead nucleic acid-guided RNA endonucleaseprotein. In some embodiments, the nucleic acid-guided RNA endonucleaseprotein is targeted to HIV RNA in a blood RNA sample, and host RNA iswashed away. In some embodiments, nucleic acid-guided RNA endonucleaseis C2c2.

In another aspect, the invention provides a composition comprising anucleic acid fragment, a nucleic acid-guided nuclease nickase-gNAcomplex, and labeled nucleotides. In one embodiment, the nucleic acidcomprises DNA. In one embodiment, the nucleic acid-guided nucleasenickase is selected from the group consisting of CAS Class I Type Inickase, CAS Class I Type III nickase, CAS Class I Type IV nickase, CASClass II Type II nickase, and CAS Class II Type V nickase. In oneembodiment, the nucleic acid-guided nuclease nickase is selected fromthe group consisting of Cas9 nickase, Cpf1 nickase, Cas3 nickase,Cas8a-c nickase, Cas10 nickase, Cse1 nickase, Csy1 nickase, Csn2nickase, Cas4 nickase, Csm2 nickase, Cm5 nickase, Csf1 nickase, C2C2nickase, and NgAgo nickase. In one embodiment, the gNAs are gRNAs. Inone embodiment, the gNAs are gDNAs. In one embodiment, the nucleic acidfragment comprises DNA. In one embodiment, the nucleic acid fragmentcomprises RNA. In one embodiment, the nucleotides are labeled withbiotin. In one embodiment, the nucleotides are part of an antibodyconjugate pair.

In another aspect, the invention provides a composition comprising anucleic acid fragment and a catalytically dead nucleic acid-guidednuclease-gNA complex, wherein the catalytically dead nucleic acid-guidednuclease is fused to a transposase. In one embodiment, thecatalytically-dead nucleic acid-guided nuclease is selected from thegroup consisting of dead CAS Class I Type I, dead CAS Class I Type III,dead CAS Class I Type IV, dead CAS Class II Type II, and dead CAS ClassII Type V. In one embodiment, the catalytically dead nucleic acid-guidednuclease is selected from the group consisting of dCas9, dCpf1, dCas3,dCas8a-c, dCas10, dCse1, dCsy1, dCsn2, dCas4, dCsm2, dCm5, dCsf1, dC2C2,and dNgAgo. In one embodiment, the gNAs are gRNAs. In one embodiment,the gNAs are gDNAs. In one embodiment, the nucleic acid fragmentcomprises DNA. In one embodiment, the nucleic acid fragment comprisesRNA. In one embodiment, the catalytically dead nucleic acid-guidednuclease is fused to the N-terminus of the transposase. In oneembodiment, the catalytically dead nucleic acid-guided nuclease is fusedto the C-terminus of the transposase. In one embodiment, the compositioncomprises a DNA fragment and a dCas9-gRNA complex, wherein the dCas9 isfused to a transposase.

In another aspect, the invention provides a composition comprising anucleic acid fragment comprising methylated nucleotides, a nucleicacid-guided nuclease nickase-gNA complex, and unmethylated nucleotides.In one embodiment, the nucleic acid-guided nuclease nickase is selectedfrom the group consisting of CAS Class I Type I nickase, CAS Class IType III nickase, CAS Class I Type IV nickase, CAS Class II Type IInickase, and CAS Class II Type V nickase. In one embodiment, the nucleicacid-guided nuclease nickase is selected from the group consisting ofCas9 nickase, Cpf1 nickase, Cas3 nickase, Cas8a-c nickase, Cas10nickase, Cse1 nickase, Csy1 nickase, Csn2 nickase, Cas4 nickase, Csm2nickase, Cm5 nickase, Csf1 nickase, C2C2 nickase, and NgAgo nickase. Inone embodiment, the gNAs are gRNAs. In one embodiment, the gNAs aregDNAs. In one embodiment, the nucleic acid fragment comprises DNA. Inone embodiment, the nucleic acid fragment comprises RNA. In oneembodiment, the nucleotides are labeled with biotin. In one embodiment,the nucleotides are part of an antibody conjugate pair. In oneembodiment, the composition comprises a DNA fragment comprisingmethylated nucleotides, a nickase Cas9-gRNA complex, and unmethylatednucleotides.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates a first protocol for the capture of target nucleicacids from a library of human genomic DNA.

FIG. 2 further illustrates the protocol for the capture of targetnucleic acids (e.g. DNA) from a nucleic acid mixture. Target nucleicacid is cut with a nucleic acid-guided nuclease, following whichadapters are ligated into the newly available blunt ends.

FIG. 3 illustrates that Cas9 cutting, followed by ligation of adapters,allows for specific amplification of target DNA.

FIG. 4 illustrates that upon sequencing the amplified DNA, ligation ofadapters occurred only at the location specified by the guide RNA. AUKsequence, after Cas9 cleavage and adapter ligation (upper left panel):SEQ ID NO: 1. Adapter sequence, after Cas9 cleavage and adapter ligation(upper right panel): SEQ ID NO: 2. AUK sequence, original sequence(lower panel: SEQ ID NO: 3. Cas9/gRNA1 binding site (at bottom): SEQ IDNO: 4.

FIG. 5 illustrates that the method of FIG. 1 efficiently amplifies DNAthat is under-represented in any given library.

FIG. 6 illustrates a second protocol for capture: the use of a nucleicacid-guided nuclease nickase to label target nucleic acids (e.g. DNA),allowing for further capture and purification.

FIG. 7 illustrates a proof of principle experiment using a restrictionnickase as a substitute for the nucleic acid-guided nuclease nickase.

FIG. 8 illustrates that enrichment of test DNA by approximately 50-foldfor the experiment illustrated in FIG. 7 (using a Cas9-nickase).

FIG. 9 illustrates a third protocol for capture: the use of acatalytically dead nucleic acid-guided nuclease-transposase fusion toinsert adaptors in human genomic library, to allowing for enrichment ofspecific SNPs.

FIG. 10 illustrates a fourth protocol for capture: the use of deadnucleic acid-guided nuclease to protect targeted sites from subsequentfragmentation by a nucleic acid-guided nuclease, allowing for enrichmentof regions of interest.

FIG. 11 illustrates a fifth protocol for capture: the use of a nucleicacid-guided nuclease nickase to protect and then enrich any targetedregion, for example SNPs or STRs, from, for example, human genomic DNA,by replacing methylated DNA with unmethylated DNA.

FIG. 12 illustrates that the methylation of test DNA in the fifthprotocol renders it susceptible to DpnI-mediated cleavage.

FIG. 13 illustrates a sixth protocol for capture: the use of a nucleicacid-guided nuclease nickase to introduce two double stranded breaksdelineating a region of interest, allowing for 3′ single strandedligation of adapters and subsequent enrichment.

DETAILED DESCRIPTION OF THE INVENTION Definitions

Unless defined otherwise herein, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Although any methodsand materials similar or equivalent to those described herein can beused in the practice or testing of the present invention, the preferredmethods and materials are described.

The headings provided herein are not limitations of the various aspectsor embodiments of the invention. Accordingly, the terms definedimmediately below are more fully defined by reference to thespecification as a whole.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Singleton, et al., DICTIONARYOF MICROBIOLOGY AND MOLECULAR BIOLOGY, 2D ED., John Wiley and Sons, NewYork (1994), and Hale & Markham, THE HARPER COLLINS DICTIONARY OFBIOLOGY, Harper Perennial, N.Y. (1991) provide one of skill with thegeneral meaning of many of the terms used herein. Still, certain termsare defined below for the sake of clarity and ease of reference.

Numeric ranges are inclusive of the numbers defining the range.

The term “sample” as used herein relates to a material or mixture ofmaterials, typically, although not necessarily, in liquid form,containing one or more analytes of interest.

The term “nucleic acid sample,” as used herein denotes a samplecontaining nucleic acids. Nucleic acid samples used herein may becomplex in that they contain multiple different molecules that containsequences. Genomic DNA from a mammal is a type of a complex sample.Complex samples may have more then 10⁴, 10⁵, 10⁶ or 10⁷ differentnucleic acid molecules. A DNA target may originate from any source suchas genomic DNA, cDNA, or an artificial DNA construct. Any samplecontaining nucleic acid, e.g., genomic DNA made from tissue culturecells or a sample of tissue, may be employed herein.

The term “nucleotide” is intended to include those moieties that containnot only the known purine and pyrimidine bases, but also otherheterocyclic bases that have been modified. Such modifications includemethylated purines or pyrimidines, acylated purines or pyrimidines,alkylated riboses or other heterocycles. In addition, the term“nucleotide” includes those moieties that contain hapten or fluorescentlabels and may contain not only conventional ribose and deoxyribosesugars, but other sugars as well. Modified nucleosides or nucleotidesalso include modifications on the sugar moiety, e.g., wherein one ormore of the hydroxyl groups are replaced with halogen atoms or aliphaticgroups, or are functionalized as ethers, amines, or the like.

The term “nucleic acids” and “polynucleotides” are used interchangeablyherein. Polynucleotide is used to describe a nucleic acid polymer of anylength, e.g., greater than about 2 bases, greater than about 10 bases,greater than about 100 bases, greater than about 500 bases, greater than1000 bases, up to about 10,000 or more bases composed of nucleotides,e.g., deoxyribonucleotides or ribonucleotides, and may be producedenzymatically or synthetically (e.g., PNA as described in U.S. Pat. No.5,948,902 and the references cited therein) which can hybridize withnaturally occurring nucleic acids in a sequence specific manneranalogous to that of two naturally occurring nucleic acids, e.g., canparticipate in Watson-Crick base pairing interactions.Naturally-occurring nucleotides include guanine, cytosine, adenine andthymine (G, C, A and T, respectively). DNA and RNA have a deoxyriboseand ribose sugar backbones, respectively, whereas PNA's backbone iscomposed of repeating N-(2-aminoethyl)-glycine units linked by peptidebonds. In PNA various purine and pyrimidine bases are linked to thebackbone by methylene carbonyl bonds. A locked nucleic acid (LNA), oftenreferred to as inaccessible RNA, is a modified RNA nucleotide. Theribose moiety of an LNA nucleotide is modified with an extra bridgeconnecting the 2′ oxygen and 4′ carbon. The bridge “locks” the ribose inthe 3′-endo (North) conformation, which is often found in the A-formduplexes. LNA nucleotides can be mixed with DNA or RNA residues in theoligonucleotide whenever desired. The term “unstructured nucleic acid,”or “UNA,” is a nucleic acid containing non-natural nucleotides that bindto each other with reduced stability. For example, an unstructurednucleic acid may contain a G′ residue and a C′ residue, where theseresidues correspond to non-naturally occurring forms, i.e., analogs, ofG and C that base pair with each other with reduced stability, butretain an ability to base pair with naturally occurring C and Gresidues, respectively. Unstructured nucleic acid is described inUS20050233340, which is incorporated by reference herein for disclosureof UNA.

The term “oligonucleotide” as used herein denotes a single-strandedmultimer of nucleotides.

Unless otherwise indicated, nucleic acids are written left to right in5′ to 3′ orientation; amino acid sequences are written left to right inamino to carboxy orientation, respectively.

The term “cleaving,” as used herein, refers to a reaction that breaksthe phosphodiester bonds between two adjacent nucleotides in bothstrands of a double-stranded DNA molecule, thereby resulting in adouble-stranded break in the DNA molecule.

The term “cleavage site, as used herein, refers to the site at which adouble-stranded DNA molecule has been cleaved.

The “nucleic acid-guided nuclease-gNA complex” refers to a complexcomprising a nucleic acid-guided nuclease protein and a guide nucleicacid (gNA, for example a gRNA or a gDNA). For example the “Cas9-gRNAcomplex” refers to a complex comprising a Cas9 protein and a guide RNA(gRNA). The nucleic acid-guided nuclease may be any type of nucleicacid-guided nuclease, including but not limited to wild type nucleicacid-guided nuclease, a catalytically dead nucleic acid-guided nuclease,or a nucleic acid-guided nuclease-nickase.

The term “nucleic acid-guided nuclease-associated guide NA” refers to aguide nucleic acid (guide NA). The nucleic acid-guidednuclease-associated guide NA may exist as an isolated nucleic acid, oras part of a nucleic acid-guided nuclease-gNA complex, for example aCas9-gRNA complex.

The terms “capture” and “enrichment” are used interchangeably herein,and refer to the process of selectively isolating a nucleic acid regioncontaining: sequences of interest, targeted sites of interest, sequencesnot of interest, or targeted sites not of interest.

The term “hybridization” refers to the process by which a strand ofnucleic acid joins with a complementary strand through base pairing asknown in the art. A nucleic acid is considered to be “selectivelyhybridizable” to a reference nucleic acid sequence if the two sequencesspecifically hybridize to one another under moderate to high stringencyhybridization and wash conditions. Moderate and high stringencyhybridization conditions are known (see, e.g., Ausubel, et al., ShortProtocols in Molecular Biology, 3rd ed., Wiley & Sons 1995 and Sambrooket al., Molecular Cloning: A Laboratory Manual, Third Edition, 2001 ColdSpring Harbor, N.Y.). One example of high stringency conditions includeshybridization at about 42° C. in 50% formamide, 5×SSC, 5×Denhardt'ssolution, 0.5% SDS and 100 μg/ml denatured carrier DNA followed bywashing two times in 2×SSC and 0.5% SDS at room temperature and twoadditional times in 0.1×SSC and 0.5% SDS at 42° C.

The term “duplex,” or “duplexed,” as used herein, describes twocomplementary polynucleotides that are base-paired, i.e., hybridizedtogether.

The term “amplifying” as used herein refers to generating one or morecopies of a target nucleic acid, using the target nucleic acid as atemplate.

The term “genomic region,” as used herein, refers to a region of agenome, e.g., an animal or plant genome such as the genome of a human,monkey, rat, fish or insect or plant. In certain cases, anoligonucleotide used in the method described herein may be designedusing a reference genomic region, i.e., a genomic region of knownnucleotide sequence, e.g., a chromosomal region whose sequence isdeposited at NCBI's Genbank database or other databases, for example.

The term “genomic sequence,” as used herein, refers to a sequence thatoccurs in a genome. Because RNAs are transcribed from a genome, thisterm encompasses sequence that exist in the nuclear genome of anorganism, as well as sequences that are present in a cDNA copy of an RNA(e.g., an mRNA) transcribed from such a genome.

The term “genomic fragment,” as used herein, refers to a region of agenome, e.g., an animal or plant genome such as the genome of a human,monkey, rat, fish or insect or plant. A genomic fragment may be anentire chromosome, or a fragment of a chromosome. A genomic fragment maybe adapter ligated (in which case it has an adapter ligated to one orboth ends of the fragment, or to at least the 5′ end of a molecule), ormay not be adapter ligated.

In certain cases, an oligonucleotide used in the method described hereinmay be designed using a reference genomic region, i.e., a genomic regionof known nucleotide sequence, e.g., a chromosomal region whose sequenceis deposited at NCBI's Genbank database or other databases, for example.Such an oligonucleotide may be employed in an assay that uses a samplecontaining a test genome, where the test genome contains a binding sitefor the oligonucleotide.

The term “ligating,” as used herein, refers to the enzymaticallycatalyzed joining of the terminal nucleotide at the 5′ end of a firstDNA molecule to the terminal nucleotide at the 3′ end of a second DNAmolecule.

If two nucleic acids are “complementary,” each base of one of thenucleic acids base pairs with corresponding nucleotides in the othernucleic acid. The term “complementary” and “perfectly complementary” areused synonymously herein.

The term “separating,” as used herein, refers to physical separation oftwo elements (e.g., by size or affinity, etc.) as well as degradation ofone element, leaving the other intact. For example, size exclusion canbe employed to separate nucleic acids, including cleaved targetedsequences.

In a cell, DNA usually exists in a double-stranded form, and as such,has two complementary strands of nucleic acid referred to herein as the“top” and “bottom” strands. In certain cases, complementary strands of achromosomal region may be referred to as “plus” and “minus” strands, the“first” and “second” strands, the “coding” and “noncoding” strands, the“Watson” and “Crick” strands or the “sense” and “antisense” strands. Theassignment of a strand as being a top or bottom strand is arbitrary anddoes not imply any particular orientation, function or structure. Untilthey become covalently linked, the first and second strands are distinctmolecules. For ease of description, the “top” and “bottom” strands of adouble-stranded nucleic acid in which the top and bottom strands havebeen covalently linked will still be described as the “top” and “bottom”strands. In other words, for the purposes of this disclosure, the topand bottom strands of a double-stranded DNA do not need to be separatedmolecules. The nucleotide sequences of the first strand of severalexemplary mammalian chromosomal regions (e.g., BACs, assemblies,chromosomes, etc.) is known, and may be found in NCBI's Genbankdatabase, for example.

The term “top strand,” as used herein, refers to either strand of anucleic acid but not both strands of a nucleic acid. When anoligonucleotide or a primer binds or anneals “only to a top strand,” itbinds to only one strand but not the other. The term “bottom strand,” asused herein, refers to the strand that is complementary to the “topstrand.” When an oligonucleotide binds or anneals “only to one strand,”it binds to only one strand, e.g., the first or second strand, but notthe other strand. If an oligonucleotide binds or anneals to both strandsof a double-stranded DNA, the oligonucleotide may have two regions, afirst region that hybridizes with the top strand of the double-strandedDNA, and a second region that hybridizes with the bottom strand of thedouble-stranded DNA.

The term “double-stranded DNA molecule” refers to both double-strandedDNA molecules in which the top and bottom strands are not covalentlylinked, as well as double-stranded DNA molecules in which the top andbottom stands are covalently linked. The top and bottom strands of adouble-stranded DNA are base paired with one other by Watson-Crickinteractions.

The term “denaturing,” as used herein, refers to the separation of atleast a portion of the base pairs of a nucleic acid duplex by placingthe duplex in suitable denaturing conditions. Denaturing conditions arewell known in the art. In one embodiment, in order to denature a nucleicacid duplex, the duplex may be exposed to a temperature that is abovethe T, of the duplex, thereby releasing one strand of the duplex fromthe other. In certain embodiments, a nucleic acid may be denatured byexposing it to a temperature of at least 90° C. for a suitable amount oftime (e.g., at least 30 seconds, up to 30 mins). In certain embodiments,fully denaturing conditions may be used to completely separate the basepairs of the duplex. In other embodiments, partially denaturingconditions (e.g., with a lower temperature than fully denaturingconditions) may be used to separate the base pairs of certain parts ofthe duplex (e.g., regions enriched for A-T base pairs may separate whileregions enriched for G-C base pairs may remain paired). Nucleic acid mayalso be denatured chemically (e.g., using urea or NaOH).

The term “genotyping,” as used herein, refers to any type of analysis ofa nucleic acid sequence, and includes sequencing, polymorphism (SNP)analysis, and analysis to identify rearrangements.

The term “sequencing,” as used herein, refers to a method by which theidentity of consecutive nucleotides of a polynucleotide are obtained.

The term “next-generation sequencing” refers to the so-calledparallelized sequencing-by-synthesis or sequencing-by-ligationplatforms, for example, those currently employed by Illumina, LifeTechnologies, and Roche, etc. Next-generation sequencing methods mayalso include nanopore sequencing methods or electronic-detection basedmethods such as Ion Torrent technology commercialized by LifeTechnologies.

The term “complementary DNA” or cDNA refers to a double-stranded DNAsample that was produced from an RNA sample by reverse transcription ofRNA (using primers such as random hexamers or oligo-dT primers) followedby second-strand synthesis by digestion of the RNA with RNaseH andsynthesis by DNA polymerase.

The term “RNA promoter adapter” is an adapter that contains a promoterfor a bacteriophage RNA polymerase, e.g., the RNA polymerase frombacteriophage T3, T7, SP6 or the like.

Other definitions of terms may appear throughout the specification.

Exemplary Methods of the Invention

As described herein, the invention provides exemplary protocols for thecapture of nucleic acids and compositions for use in these protocols.Exemplary protocols are illustrated in FIGS. 1, 6, 9, 10, 11, and 13,respectively, and in the Examples section. Various uses are contemplatedthroughout. Specific terms referred to in this section are described ingreater detail in subsequent sections.

In one embodiment, the invention provides a capture method (depicted asProtocol 1) as provided in FIGS. 1-5. In this embodiment, the method isused to capture target nucleic acid sequences. Referring to FIG. 1, themethod comprises providing a sample or a library 100, subject toextraction protocols 101 (e.g. DNA extraction protocols), resulting in asample 102 comprising >99% non-target sequence and <1% target sequence.The sample is subjected to library construction protocols 103, resultingin a nucleic acid library comprising sequencing indexed adapters 104,resulting in a plurality of adapter-ligated nucleic acids, wherein thenucleic acids are ligated to a first adapter at one end, and a secondadapter at the other end. To produce a library of target-specific guideNAs (e.g. gRNAs), a library of target specific gNA precursors 110, eachcomprising a RNA polymerase promoter 111, a specific-base pair region112 (e.g. a 20 base pair region), and a stem-loop binding site for anucleic acid-guided nuclease 113, was subjected to in vitrotranscription 114, yielding a library of target-specific guide RNAs 115.The library of target-specific guide NAs was then combined with nucleicacid-guided nuclease proteins 116 to yield a library of nucleicacid-guided nuclease-gNA complexes. The nucleic acid-guided nuclease-gNAcomplexes were then combined with the nucleic acid library such that thenucleic acid-guided nuclease cleaved matching target nucleic acidsequences and left other nucleic acids uncleaved 117. Second adapters118 were added and allowed to ligate specifically to the 5′phosphorylated blunt ends of cleaved nucleic acids 119. This allows fordownstream applications, for example amplifying the nucleic acidfragments comprising a first or second adapter at one end and a thirdadapter at the other end, using adapter-specific PCR 120.

In an exemplary depiction of Protocol 1, referring to FIG. 1, the methodis used to capture target nucleic acid sequences. The method comprisesproviding a sample or a library comprising a plurality ofadapter-ligated nucleic acids, wherein the nucleic acids are ligated toa first adapter at one end, and a second adapter at the other end. Thisis followed by contacting the sample with a plurality of Cas9-gRNAcomplexes, wherein the gRNAs are complementary to targeted sites ofinterest contained in a subset of the nucleic acids. The contactingcleaves the targeted sites of interest, thereby generating a pluralityof nucleic acid fragments ligated to a first or second adapter at oneend and no adapter at the other end. This step is followed by ligatingthe plurality of resulting nucleic acid fragments with third adapters,thereby generating a plurality of nucleic acid fragments ligated to afirst or second adapter at one end and a third adapter at the other end.This allows for downstream applications, for example amplifying thenucleic acid fragments comprising a first or second adapter at one endand a third adapter at the other end, using adapter-specific PCR.

In one embodiment, the invention provides a capture method (depicted asProtocol 2) as provided in FIGS. 6-8. In this embodiment, the method isused to introduce labeled nucleotides at targeted sites of interest. Themethod comprises providing a sample comprising a plurality of doublestranded nucleic acid fragments 601 (e.g. double stranded DNA);contacting the sample with a plurality of nucleic acid-guided nucleasenickase-gNA complexes. The nickase nucleic acid-guided nuclease 603 isguided by target-specific guide NAs 604, wherein the gNAs arecomplementary to targeted sites of interest in the nucleic acidfragments, thereby generating a plurality of nicked nucleic acidfragments at the targeted sites of interest. Nickase is used to nicktarget sequences 605. The nickase-nucleic acid-guided nuclease cleavesat target sequence. Single strand cuts (nicks) are a substrate for DNApolymerase I which can be used to replace DNA downstream of the nickwith biotin labeled DNA 606.

In an exemplary depiction of Protocol 2, referring to FIG. 6, the methodis used to introduce labeled nucleotides at targeted sites of interest.The method comprises providing a sample comprising a plurality ofnucleic acid fragments; contacting the sample with a plurality of Cas9nickase-gRNA complexes, wherein the gRNAs are complementary to targetedsites of interest in the nucleic acid fragments, thereby generating aplurality of nicked nucleic acid fragments at the targeted sites ofinterest; and then is followed by contacting the plurality of nickednucleic acid fragments with an enzyme capable of initiating nucleic acidsynthesis at a nicked site, and labeled nucleotides, thereby generatinga plurality of nucleic acid fragments comprising labeled nucleotides inthe targeted sites of interest.

In one embodiment, the invention provides a capture method (depicted asProtocol 3) as provided in FIG. 9. In this embodiment, the method isused to capture target nucleic acid sequences of interest 901. Themethod first involves providing a sample comprising a plurality ofadapter-ligated nucleic acids 902, wherein the nucleic acids are ligatedto a first adapter at one end and are ligated to a second adapter at theother end. This is then followed by contacting the sample with aplurality of catalytically dead nucleic acid-guided nuclease-gNAcomplexes, wherein the catalytically dead nucleic acid-guided nucleaseis fused to a transposase 903, wherein the gNAs 904 are complementary totargeted sites of interest contained in a subset of the nucleic acids,and wherein the catalytically dead nucleic acid-guided nuclease-gNAtransposase complexes are loaded with a plurality of third adapters 905,to generate a plurality of nucleic acids fragments 906 comprising eithera first or second adapter at one end and a third adapter at the otherend. These fragments can then be amplified 907 using the adaptersequences and then sequenced 908.

In an exemplary depiction of Protocol 3, referring to FIG. 9, the methodis used to capture target nucleic acid sequences of interest. The methodfirst involves providing a sample comprising a plurality ofadapter-ligated nucleic acids, wherein the nucleic acids are ligated toa first adapter at one end and are ligated to a second adapter at theother end. This is then followed by contacting the sample with aplurality of dCas9-gRNA complexes, wherein the dCas9 is fused to atransposase, wherein the gRNAs are complementary to targeted sites ofinterest contained in a subset of the nucleic acids, and wherein thedCas9-gRNA transposase complexes are loaded with a plurality of thirdadapters, to generate a plurality of nucleic acids fragments comprisingeither a first or second adapter at one end and a third adapter at theother end.

In one embodiment, the invention provides a capture method (depicted asProtocol 4) as provided for example in FIG. 10. In this embodiment, themethod is used to capture target nucleic acid sequences of interest1001. The method comprises first providing a sample comprising aplurality of adapter-ligated nucleic acids 1002, wherein the nucleicacids are ligated to the adapter at the 5′ end and 3′ ends. The methodthen involves contacting the sample with a plurality of catalyticallydead nucleic acid-guided nuclease-gNA complexes 1003, wherein the gNAs1004 are complementary to targeted sites of interest contained in asubset of the nucleic acids, thereby generating a plurality of nucleicacids adapter-ligated at the 5′ and 3′ ends, bound to a catalyticallydead nucleic acid-guided nuclease-gNA complex 1005. This is followed bycontacting the sample with a plurality of nucleic acid-guidednuclease-gNA complexes 1006, wherein the gNAs 1007 are complementary totargeted sites of interest and targeted sites not of interest in thenucleic acids, thereby generating a plurality of nucleic acid fragments1008 comprising nucleic acid sequences not of interest, adapter ligatedat only one of the 5′ or 3′ ends. In this method wherein the secondcontacting step, contacting with a plurality of nucleic acid-guidednuclease-gNA complexes, does not displace the plurality of nucleic acidsadapter-ligated at the 5′ and 3′ ends, bound to a catalytically deadnucleic acid-guided nuclease-gNA complex of step (b).

In an exemplary depiction of Protocol 4, referring to FIG. 10, themethod is used to capture target nucleic acid sequences of interest. Themethod comprises first providing a sample comprising a plurality ofadapter-ligated nucleic acids, wherein the nucleic acids are ligated tothe adapter at the 5′ end and 3′ ends. The method then involvescontacting the sample with a plurality of dead nucleic acid-guidednuclease-gNA complexes (e.g., dCas9-gRNA) complexes, wherein the gNAsare complementary to targeted sites of interest contained in a subset ofthe nucleic acids, thereby generating a plurality of nucleic acidsadapter-ligated at the 5′ and 3′ ends, bound to a dead nucleicacid-guided nuclease-gNA complex (e.g., dCAS9-gRNA complex). This isfollowed by contacting the sample with a plurality of nucleicacid-guided nuclease-gNA complexes (e.g., Cas9-gRNA complexes), whereinthe gNAs are complementary to targeted sites of interest and targetedsites not of interest in the nucleic acids, thereby generating aplurality of nucleic acid fragments comprising nucleic acid sequencesnot of interest, adapter ligated at only one of the 5′ or 3′ ends. Inthis method wherein the second contacting step, contacting with aplurality of nucleic acid-guided nuclease-gNA complexes (e.g., Cas9-gRNAcomplexes), does not displace the plurality of nucleic acidsadapter-ligated at the 5′ and 3′ ends, bound to a dead nucleicacid-guided nuclease-gNA complex (e.g., dCAS9-gRNA complex) of step (b).

In one embodiment, the invention provides a capture method (depicted asProtocol 5) as provided, for example, in FIG. 11 and FIG. 12. In thisembodiment, the method is used to capture target nucleic sequences ofinterest 1101. The method involves first providing a sample comprising aplurality of sequences 1102, wherein the sequences comprise methylatednucleotides (e.g., treated with Dam methyltransferase), and wherein thesequences are adapter ligated on the 5′ and 3′ ends. The method theninvolves first contacting the sample with a plurality of nucleicacid-guided nuclease nickase-gNA complexes 1103, wherein the gNAs 1104are complementary to targeted sites of interest in a subset of thesequences, thereby generating a plurality of nicked nucleic acidsequences 1105 at the targeted sites of interest, and wherein thenucleic acid sequences are adapter ligated on the 5′ and 3′ ends. Wherethe nucleic acid is DNA, single strand cuts (nicks) can be, for example,a substrate for DNA polymerase I, which replaces the DNA downstream ofthe nick with unmethylated DNA. This can then be followed by thencontacting the sample with an enzyme capable of initiating DNA synthesisat a nicked site, and unmethylated nucleotides, thereby generating aplurality of DNA comprising unmethylated nucleotides 1106 in thetargeted sites of interest and wherein the DNA sequences are adapterligated on the 5′ and 3′ ends. This is then followed by contacting thesample with an enzyme capable of cutting methylated DNA (e.g., DpnI)1107, thereby generating a plurality of DNA fragments comprisingmethylated DNA, wherein the plurality of DNA fragments comprisingmethylated DNA are adapter ligated only one of the 5′ and 3′ ends.Remaining intact nucleic acid can be amplified and sequenced 1108. FIG.12 shows, for example, results from an experiment conducted according tothis protocol. The first column on the gel shows a 1 kb ladder, thesecond column shows test DNA treated with Dam methyltransferase, thendigested with DpnI, and the third column shows test DNA digested withDpnI. The second column shows a band corresponding to DpnI digested DNA,while the third column shows a band corresponding to uncut test DNA.

In an exemplary depiction of Protocol 5, referring to FIGS. 11-12, themethod is used to capture target DNA sequences of interest. The methodinvolves first providing a sample comprising a plurality of DNAsequences, wherein the DNA sequences comprise methylated nucleotides,and wherein the DNA sequences are adapter ligated on the 5′ and 3′ ends.The method then involves first contacting the sample with a plurality ofCas9 nickase-gRNA complexes, wherein the gRNAs are complementary totargeted sites of interest in a subset of the DNA sequences, therebygenerating a plurality of nicked DNA at the targeted sites of interest,and wherein the DNA are adapter ligated on the 5′ and 3′ ends. This isthen followed by then contacting the sample with an enzyme capable ofinitiating DNA synthesis at a nicked site, and unmethylated nucleotides,thereby generating a plurality of DNA comprising unmethylatednucleotides in the targeted sites of interest and wherein the DNAsequences are adapter ligated on the 5′ and 3′ ends. This is thenfollowed by contacting the sample with an enzyme capable of cuttingmethylated DNA, thereby generating a plurality of DNA fragmentscomprising methylated DNA, wherein the plurality of DNA fragmentscomprising methylated DNA are adapter ligated only one of the 5′ and 3′ends.

In one embodiment, the invention provides a capture method (depicted asProtocol 6) as provided in FIG. 13. The objective of this method is toenrich a region of a nucleic acid 1301, from any source (e.g., library1302, genomic, or PCR), as depicted for example in FIG. 13. The nucleicacid-guided nuclease-nickase 1303 can be targeted to proximal sitesusing two guide NAs 1304 and 1305, resulting in nicking of nucleic acidat each location 1306. Alternatively, an adapter can be ligated on onlyone side, then filled in, then an adapter can be ligated on the otherside. The two nicks can be close to each other (e.g., within 10 to 15bp). Single nicks may be generated in non-target molecules. Because ofthe proximity of the two nicking sites, a double stranded break can becreated when the reaction is heated 1307, e.g. to 65° C., resulting inlong (e.g., 10-15 bp) 3′ overhangs. These overhangs can be recognized bya thermostable single stranded DNA/RNA ligase, to allow forsite-specific ligation 1308 of single stranded adapters. The ligase can,for example, only recognize long 3′ overhangs, thus ensuring thatadapters will not be ligated at other sites. This process can berepeated using nucleic acid-guided nuclease nickase and guide NAstargeting on the other side of the region of interest, followed byligation as above using a second single stranded adapter. Once twoadapters have been ligated on either side of the region of interest, theregion can be amplified or sequenced directly 1309.

In an exemplary depiction of Protocol 6, referring to FIG. 13, themethod is to enrich a region of DNA, from any DNA source (e.g., library,genomic, or PCR). A Cas9 Nickase can be targeted to proximal sites usingtwo guide RNAs and, resulting in nicking of DNA at each location.Alternatively, an adapter can be ligated on only one side, then filledin, then an adapter can be ligated on the other side. The two nicks canbe close to each other (e.g., within 10 to 15 bp). Single nicks may begenerated in non-target molecules. Because of the proximity of the twonicking sites, a double stranded break can be created when the reactionis heated, e.g. to 65° C., resulting in long (e.g., 10-15 bp) 3′overhangs. These overhangs can be recognized by a thermostable singlestranded DNA/RNA ligase, such as a Thermostable 5′App DNA/RNA ligase toallow for site-specific ligation of single stranded adapters. The ligasecan, for example, only recognize long 3′ overhangs, thus ensuring thatadapters will not be ligated at other sites. This process can berepeated using Cas9 Nickase and guide RNA targeting on the other side ofthe region of interest, followed by ligation as above using a secondsingle stranded adapter. Once two adapters have been ligated on eitherside of the region of interest, the region can be amplified or sequenceddirectly.

In one embodiment, provided herein is a method of enriching a sample forsequences of interest, comprising: (a) providing a sample comprisingsequences of interest and targeted sequences for depletion, wherein thesequences of interest comprise less than 50% of the sample; and (b)contacting the sample with a plurality of nucleic acid-guided RNAendonuclease-gRNA complexes or a plurality of nucleic acid-guided DNAendonuclease-gDNA complexes, wherein the gRNAs and gDNAs arecomplementary to the targeted sequences. In some embodiments, thetargeted sequences are thereby cleaved. In one embodiment, the nucleicacid-guided RNA endonuclease is C2c2. In one embodiment the C2c2 iscatalytically dead. In one embodiment, the nucleic acid-guided DNAendonuclease is NgAgo (Argonaute from Natronobacterium gregoryi). In oneembodiment the NgAgo is catalytically dead.

In one embodiment, provided herein is a method of enriching a samplecomprising: (a) providing a sample comprising host nucleic acids andnon-host nucleic acids; (b) contacting the sample with a plurality ofnucleic acid-guided RNA endonuclease-gRNA complexes or plurality ofnucleic acid-guided DNA endonuclease-gDNA complexes, wherein the gNAsare complementary to targeted sites in the host nucleic acids, and (c)enriching the sample for non-host nucleic acids. In one embodiment, thenucleic acid-guided RNA endonuclease is C2c2. In one embodiment the C2c2is catalytically dead. In one embodiment, the nucleic acid-guided DNAendonuclease is NgAgo. In one embodiment the NgAgo is catalyticallydead.

Nucleic Acids, Samples

Nucleic acids of the invention (targeted for capture) can be any DNA,any RNA, single stranded DNA, single stranded RNA, double stranded DNA,double stranded RNA, artificial DNA, artificial RNA, synthetic DNA,synthetic RNA, and RNA/DNA hybrids.

The nucleic acids of the invention can be a genomic fragment, comprisinga region of the genome, or the whole genome itself. In one embodiment,the genome is a DNA genome. In another embodiment, the genome is a RNAgenome.

Nucleic acids of the invention can be obtained from a eukaryotic orprokaryotic organism; from a mammalian organism or a non-mammalianorganism; from an animal or a plant; from a bacteria or virus; from ananimal parasite; or from a pathogen.

Nucleic acids of the invention can be obtained from any mammalianorganism. In one embodiment the mammal is a human. In another embodimentthe mammal is a livestock animal, for example a horse, a sheep, a cow, apig, or a donkey. In another embodiment, a mammalian organism is adomestic pet, for example a cat, a dog, a gerbil, a mouse, a rat. Inanother embodiment the mammal is a type of a monkey.

Nucleic acids of the invention can be obtained from any bird or avianorganism. An avian organism includes but is not limited to chicken,turkey, duck and goose.

Nucleic acids of the invention can be obtained from a plant. In oneembodiment, the plant is rice, maize, wheat, rose, grape, coffee, fruit,tomato, potato, or cotton.

In some embodiments, nucleic acids of the invention are obtained from aspecies of bacteria. In one embodiment, the bacteria aretuberculosis-causing bacteria.

In some embodiments, nucleic acids of the invention are obtained from avirus.

In some embodiments, nucleic acids of the invention are obtained from aspecies of fungi.

In some embodiments, nucleic acids of the invention are obtained from aspecies of algae.

In some embodiments, nucleic acids of the invention are obtained fromany mammalian parasite.

In some embodiments, nucleic acids of the invention are obtained fromany mammalian parasite. In one embodiment, the parasite is a worm. Inanother embodiment, the parasite is a malaria-causing parasite. Inanother embodiment, the parasite is a Leishmaniasis-causing parasite. Inanother embodiment, the parasite is an amoeba.

In some embodiments the pathogen is a non-mammalian pathogen (ispathogenic in non-mammalian organisms).

In one embodiment, the nucleic acids of the invention include nucleicacids that are targets of gNAs and nucleic acids that are not thetargets of gNAs, in the same sample.

In one embodiment, the nucleic acids of the invention include nucleicacids that are targets of gRNAs and nucleic acids that are not thetargets of gRNAs, in the same sample.

In one embodiment, the nucleic acids of the invention include nucleicacids that are targets of gDNAs and nucleic acids that are not thetargets of gDNAs, in the same sample.

In one embodiment, the nucleic acids of the invention include targetnucleic acids (targets of gNAs) and nucleic acids of interest (nottargeted by gNAs) from a sample.

In one embodiment, the nucleic acids of the invention include targetnucleic acids (targets of gRNAs) and nucleic acids of interest (nottargeted by gRNAs) from a sample.

In one embodiment, the nucleic acids of the invention include targetnucleic acids (targets of gDNAs) and nucleic acids of interest (nottargeted by gDNAs) from a sample.

In one embodiment, the target DNA (target of the gNAs, gRNAs, gDNAs) maybe human non-mitochondrial DNA (e.g. genomic DNA), and the DNA ofinterest (for capture) may be the human mitochondrial DNA, and the humanmitochondrial DNA is enriched by targeting the non-mitochondrial humanDNA.

In one embodiment, the nucleic acids to be captured may be a non-mapableregion of a genome; and the nucleic acids to be retained for furtheranalysis/sequencing/cloning may be mapable regions of a genome. In oneembodiment, the nucleic acids to be captured out may be a mapable regionof a genome; and the nucleic acids to be retained for furtheranalysis/sequencing/cloning may be non-mapable regions of a genome.Examples of non mapable regions include telomeres, centromeres, or othergenomic regions that contain features harder to map.

In one embodiment, the nucleic acids of the invention are obtained froma biological sample. The biological sample from which the nucleic acidsare obtained include but are not limited to whole blood, plasma, serum,tears, saliva, mucous, cerebrospinal fluid, teeth, bone, fingernails,feces, urine, tissue, and biopsy. The biological sample may includeforensic samples such as teeth, bone, fingernails or the like. Thebiological sample may include tissue, a tissue biopsy, for example aresected lung tissue. The biological sample may include a clinicalsample, which refers to a sample obtained in a clinical setting, such asin a hospital, or clinic.

In one embodiment, the nucleic acids of the invention are obtained froman environmental sample, for example from water, soil, air, or rock.

In one embodiment, the nucleic acids of the invention are obtained froma forensic sample, for example, a sample obtained from an individual ata crime scene, from a piece of evidence, post-mortem, as a part of anongoing investigation or the like.

In on embodiment, the nucleic acids of the invention are provided in alibrary.

The nucleic acids of the invention can be either provided or extractedfrom a sample. Extraction can extract substantially all the nucleic acidsequences from a specimen.

The methods of the invention may produce nucleic acids to be captured:nucleic acids to not be captured at a ratio of anywhere between99.999:0.001 to 0.001:99.999. The methods of the invention may producetargeted nucleic acids and nucleic acids of interest at a ratio ofanywhere between 99.999:0.001 to 0.001:99.999. The methods of theinvention may produce nucleic acids to be captured to nucleic acids tobe retained/analyzed/sequenced at a ratio of anywhere between99.999:0.001 to 0.001:99.999. In these embodiments, the ratios can beequal to or fall anywhere in between 99.999:0.001 to 0.001:99.999, forexample the ratio can be 99:1, 95:5, 90:10, 85:15, 80:20, 75:25, 70:30,65:35, 60:40, 55:45, 50:50, 45:55, 40:60, 35:65, 30:70, 25:75, 20:80,15:85, 10:90, 5:95, and 1:99.

After the capture, the captured or retained nucleic sequences can befragmented to reduce the lengths of each extracted nucleic acids to amore manageable length for amplifying, sequencing or the like.

As provided herein, at least 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%,70%, 80%, 90% of the starting nucleic acid material can be captured.This capture can be achieved in no greater than 10 minutes, 15 minutes,20 minutes, 30 minutes, 45 minutes, 60 minutes, 75 minutes, 90 minutes,105 minutes, 120 minutes, 150 minutes, 180 minutes, or 240 minutes.

In some cases, the targeted sites of interest represent less than 90%,less than 80%, less than 70%, less than 60%, less than 50%, less than40%, less than 30%, less than 20%, or less than 10% of the total DNA inthe sample.

Adapters

As provided herein, the nucleic acids of the invention (referred tointerchangeably as nucleic acids or nucleic acid fragments) areadapter-ligated, to aid in carrying out the methods provided herein.

Nucleic acids of the invention to be adapter-ligated can range from 20bp in size to 5000 bp in size. For example, the nucleic acid to beadapter-ligated may be at least 20, 25, 50, 75, 100, 125, 150, 175, 200,25, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, or 5000 bp. In onespecific embodiment, the nucleic acid to be adapter ligated is 100 bp.In one specific embodiment, the nucleic acid to be adapter ligated is200 bp. In one specific embodiment, the nucleic acid to be adapterligated is 300 bp. In one specific embodiment, the nucleic acid to beadapter ligated is 400 bp. In one specific embodiment, the nucleic acidto be adapter ligated is 500 bp.

An adapter can be ligated to each end of each of the nucleic acids, ornucleic acid fragments, at the 5′ and 3′ ends. In other embodiments anadapter may be ligated to only one end of each of the fragments or inother instances adapters may be ligated in a later step. In one examplethe adapter is a nucleic acid that is ligatable to both strands of adouble-stranded DNA molecule. In various embodiments the adapter may bea hairpin adapter e.g., one molecule that base pairs with itself to forma structure that has a double-stranded stem and a loop, where the 3′ and5′ ends of the molecule ligate to the 5′ and 3′ ends of thedouble-stranded DNA molecule of the fragment, respectively. Alternately,the adapter may be a Y-adapter ligated to one end or to both ends of afragment, also called a universal adapter. Alternately, the adapter mayitself be composed of two distinct oligonucleotide molecules that arebase paired with one another. Additionally, a ligatable end of theadapter may be designed to be compatible with overhangs made by cleavageby a restriction enzyme, or it may have blunt ends or a 5′ T overhang.Generally, the adapter may include double-stranded as well assingle-stranded molecules. Thus the adapter can be DNA or RNA, or amixture of the two. Adapters containing RNA may be cleavable by RNasetreatment or by alkaline hydrolysis.

Adapters can be 10 to 100 bp in length although adapters outside of thisrange are usable without deviating from the present invention. Inspecific embodiments, the adapter is at least 10 bp, at least 15 bp, atleast 20 bp, at least 25 bp, at least 30 bp, at least 35 bp, at least 40bp, at least 45 bp, at least 50 bp, at least 55 bp, at least 60 bp, atleast 65 bp, at least 70 bp, at least 75 bp, at least 80 bp, at least 85bp, at least 90 bp, or at least 95 bp in length.

In further examples the captured nucleic acid sequences may be derivedfrom one or more DNA sequencing libraries. An adapter may be configuredfor a next generation sequencing platform, for example for use on anIllumina sequencing platform or for use on an IonTorrent platform.

An adapter may contain a restriction site of interest or a primerbinding site.

Exemplary adapters include P5 and P7 adapters.

Guide Nucleic Acids (gNAs)

Provided herein are guide nucleic acids (gNAs), wherein the gNAs arecomplementary to (selective for, can hybridize with) targeted sites orsequences of interest, or sequences not of interest in the nucleicacids, for example in genomic DNA from a host. The gNAs guide nucleicacid-guided nucleases to specific sites on a nucleic acid.

In some embodiments, the gNAs are guide RNAs (gRNAs); in otherembodiments, the gNAs are guide DNAs (gDNAs). In some embodiments thegNAs comprise a mixture of gRNAs and gDNAs.

The host to which the gNAs are directed to can be an animal, for examplea human, cow, horse, sheep, pig, monkey, dog, cat, gerbil, bird, mouse,or rat. The host can be a plant. The non-host can be a prokaryoticorganism, a eukaryote, a virus, a bacterium, a fungus, and a protozoan.

In one embodiment, the present invention provides a guide nucleic acid(gNA) library which comprises a collection of gNAs, configured tohybridize with a nucleic acid sequence targeted for capture. In anotherembodiment, the present invention provides a guide NA library whichcomprises a collection of gNAs, configured to hybridize with a nucleicacid sequence that is not targeted for capture.

In one embodiment, the present invention provides a guide RNA librarywhich comprises a collection of gRNAs, configured to hybridize with anucleic acid sequence targeted for capture. In another embodiment, thepresent invention provides a guide RNA library which comprises acollection of gRNAs, configured to hybridize with a nucleic acidsequence that is not targeted for capture.

In one embodiment, the present invention provides a guide DNA librarywhich comprises a collection of gDNAs, configured to hybridize with anucleic acid sequence targeted for capture. In another embodiment, thepresent invention provides a guide DNA library which comprises acollection of gDNAs, configured to hybridize with a nucleic acidsequence that is not targeted for capture.

In one embodiment, the gNAs are selective for target nucleic acids in asample, but are not selective for sequences of interest from the sample.

In one embodiment, the gNAs are used to serially capture nucleic acidsequences.

In some embodiments, the gNAs are selective for a target nucleic acidsequences which are followed by Protospacer Adjacent Motif (PAM)sequences that can be bound by a nucleic acid-guided nuclease. In someembodiments, the sequence of the gNAs is determined by the nucleicacid-guided nuclease type. In various embodiments the gNAs may betailored to different nucleic acid-guided nuclease types as the PAMsequence can vary by the species of the organism from which the nucleicacid-guided nuclease is derived.

The gNAs (gRNAs or gDNAs) of the invention can range in size from 50-200base pairs. For example, a gNA of the invention can be at least 50 bp,55 bp, 60 bp, 65 bp, 70 bp, 75 bp, 80 bp, 85 bp, 90 bp, 95 bp, 100 bp,110 bp, 120 bp, 125 bp, 130 bp, 140 bp, 150 bp, 160 bp, 170 bp, 175 bp,180 bp, 190 bp, or 195 bp. In specific embodiments, the gNA is 80 bp, 90bp, 100 bp, or 110 bp. In some embodiments, a target-specific gNAcomprises a base pair sequence can be complementary to a pre-definedsite in a target nucleic acid that is followed by a Protospacer AdjacentMotif or (PAM) sequence that can be bound by a nucleic acid-guidednuclease protein (e.g. Cas9) derived from a bacterial species. Inspecific embodiments, the base pair sequence of the gNA that iscomplementary to a pre-defined site in a target nucleic acid is 15, 16,17, 18, 19, 20, 25, 30, 35, 40, 45, or 50 base pairs.

The present invention also provides for gNA libraries (e.g. gRNAlibraries or gDNA libraries). A gNA library can comprise a number ofdifferent species-specific guide NA (e.g. gRNA or gDNA) elements each,configured to hybridize with (be selective for) a nucleic acid sequencebeing targeted capture, a nucleic acid sequence of interest, or anucleic acid sequence not of interest. Each gNA includes atarget-specific guide sequence and a stem loop binding site that isformed to bind with a nucleic acid-guided nuclease protein. In someembodiments, the library can comprise a plurality of different guideNAs, each having a different 15-30 base pair sequence that iscomplementary to a different pre-defined site in the nucleic acid beingtargeted, that is followed by an appropriate PAM sequence that can bebound by a nucleic acid-guided nuclease protein. For each guide NA thePAM sequence is present in the pre-defined DNA or RNA target sequence ofthe nucleic acid of interest but is not present in the correspondingtarget specific guide sequence.

Generally according to the present invention, any nucleic acid sequencein a genome of interest, with a pre-defined target sequence followed bythe appropriate PAM sequence can be hybridized by a corresponding guideRNA provided in the guide NA library and bound by a nucleic acid-guidednuclease. In various embodiments the gNA library may be tailored todifferent nucleic acid-guided nuclease types since the PAM sequence canvary by the species of the bacteria from which nucleic acid-guidednuclease is derived. But in some variations, the pre-defined targetsequence is not followed by a PAM sequence.

Different target-specific sequences in the gNAs can be generated. Thiscan be done by using a promoter for a bacteriophage RNA polymerase,e.g., the RNA polymerase from bacteriophage T3, T7, SP6 or the like.Accordingly, each different T7 RNA polymerase promoter provides adifferent target specific sequence suitable for hybridizing to adifferent target nucleic acid sequence. A non-limiting exemplary set offorward primers usable for both annealing and subsequent PCR reactionsis listed in Table 1 provided below.

A gNA library (e.g. a gRNA or gDNA library) can be amplified to includea large number of copies of each different guide NA element as well as alarge number of different guide NA elements as may be suitable to forthe desired capture results. The number of unique guide NA elements in agiven guide NA library may range from 1 unique guide NA element to asmany as 300,000,000 unique guide NA elements, or approximately 1 uniqueguide NA sequence for every 10 base pairs in the human genome. Thenumber of unique gNAs (e.g., gRNAs or gDNAs) can be at least about 101,102, 103, 104, 105, 106, 107, or 108 unique gNAs. The number of uniquegNAs can result in that number of unique nucleic acid-guidednuclease-gNA complexes (e.g. CRISPR/Cas system protein-gRNA complexes).

Without being limited to theory, the distance between gNAs to arriveat >95% cleavage of the target nucleic acid can be computed, if the gNAsdisplay ˜100% efficacy: this can be computed by measuring thedistribution of library size and determining the mean, N and thestandard deviation SD; N−2SD=minimum size for >95% of the library,ensuring that there is one guide NA per fragment of this size toensure >95% capture. This can also be described as the Maximum distancebetween guide NAs=Mean of library size−2×(standard deviation of librarysize).

In various embodiments of the invention, the gNAs can be specific forvarious targeted sites of interest, including, but not limited to,single nucleotide polymorphisms (SNPs), short tandem repeats (STRs),cancer genes, inserts, deletions, structural variations, exons, geneticmutations, and regulatory regions.

Nucleic Acid-Guided Nucleases

Provided herein compositions and methods for the capture of nucleicacids from a sample. These compositions and methods utilize nucleicacid-guided nucleases. As used herein, a “nucleic acid-guided nuclease”is any endonuclease that cleaves DNA, RNA or DNA/RNA hybrids, and whichuses one or more nucleic acid guide nucleic acids (gNAs) to conferspecificity. Nucleic acid-guided nucleases include CRISPR/Cas systemproteins as well as non-CRISPR/Cas system proteins.

The nucleic acid-guided nucleases provided herein can be DNA guided DNAendonucleases; DNA guided RNA endonucleases; RNA guided DNAendonucleases; or RNA guided RNA endonucleases.

In one embodiment, the nucleic acid-guided nuclease is a nucleicacid-guided-DNA endonuclease.

In one embodiment, the nucleic acid-guided nuclease is a nucleicacid-guided-RNA endonuclease.

CRISPR/Cas System Nucleic Acid-Guided Nucleases

In some embodiments, CRISPR/Cas system proteins are used in theembodiments provided herein. In some embodiments, CRISPR/Cas systemproteins include proteins from CRISPR Type I systems, CRISPR Type IIsystems, and CRISPR Type III systems.

In some embodiments, CRISPR/Cas system proteins can be from anybacterial or archaeal species.

In some embodiments, the CRISPR/Cas system protein is isolated,recombinantly produced, or synthetic.

In some embodiments, the CRIPR/Cas system proteins are from, or arederived from CRISPR/Cas system proteins from Streptococcus pyogenes,Staphylococcus aureus, Neisseria meningitidis, Streptococcusthermophiles, Treponema denticola, Francisella tularensis, Pasteurellamultocida, Campylobacter jejuni, Campylobacter lari, Mycoplasmagallisepticum, Nitratifractor salsuginis, Parvibaculum lavamentivorans,Roseburia intestinalis, Neisseria cinerea, Gluconacetobacterdiazotrophicus, Azospirillum, Sphaerochaeta globus, Flavobacteriumcolumnare, Fluviicola taffensis, Bacteroides coprophilus, Mycoplasmamobile, Lactobacillus farciminis, Streptococcus pasteurianus,Lactobacillus johnsonii, Staphylococcus pseudintermedius, Filifactoralocis, Legionella pneumophila, Suterella wadsworthensis, orCorynebacter diphtheria.

In some embodiments, examples of CRISPR/Cas system proteins can benaturally occurring or engineered versions.

In some embodiments, naturally occurring CRISPR/Cas system proteins canbelong to CAS Class I Type I, III, or IV, or CAS Class II Type II or V,and can include Cas9, Cas3, Cas8a-c, Cas10, Cse1, Csy1, Csn2, Cas4,Csm2, Cmr5, Csf1, C2c2, and Cpf1.

In an exemplary embodiment, the CRISPR/Cas system protein comprisesCas9.

A “CRISPR/Cas system protein-gNA complex” refers to a complex comprisinga CRISPR/Cas system protein and a guide NA (e.g. a gRNA or a gDNA).Where the gNA is a gRNA, the gRNA may be composed of two molecules,i.e., one RNA (“crRNA”) which hybridizes to a target and providessequence specificity, and one RNA, the “tracrRNA”, which is capable ofhybridizing to the crRNA. Alternatively, the guide RNA may be a singlemolecule (i.e., a gRNA) that contains crRNA and tracrRNA sequences.

A CRISPR/Cas system protein may be at least 60% identical (e.g., atleast 70%, at least 80%, or 90% identical, at least 95% identical or atleast 98% identical or at least 99% identical) to a wild type CRISPR/Cassystem protein. The CRISPR/Cas system protein may have all the functionsof a wild type CRISPR/Cas system protein, or only one or some of thefunctions, including binding activity, nuclease activity, and nucleaseactivity.

The term “CRISPR/Cas system protein-associated guide NA” refers to aguide NA. The CRISPR/Cas system protein-associated guide NA may exist asisolated NA, or as part of a CRISPR/Cas system protein-gNA complex.

Cas9

In some embodiments the CRISPR/Cas System protein nucleic acid-guidednuclease is or comprises Cas9. The Cas9 of the present invention can beisolated, recombinantly produced, or synthetic.

Examples of Cas9 proteins that can be used in the embodiments herein canbe found in F. A. Ran, L. Cong, W. X. Yan, D. A. Scott, J. S.Gootenberg, A. J. Kriz, B. Zetsche, O. Shalem, X. Wu, K. S. Makarova, E.V. Koonin, P. A. Sharp, and F. Zhang; “In vivo genome editing usingStaphylococcus aureus Cas9,” Nature 520, 186-191 (9 Apr. 2015)doi:10.1038/nature14299, which is incorporated herein by reference.

In some embodiments, the Cas9 is a Type II CRISPR system derived fromStreptococcus pyogenes, Staphylococcus aureus, Neisseria meningitidis,Streptococcus thermophiles, Treponema denticola, Francisella tularensis,Pasteurella multocida, Campylobacter jejuni, Campylobacter lari,Mycoplasma gallisepticum, Nitratifractor salsuginis, Parvibaculumlavamentivorans, Roseburia intestinalis, Neisseria cinerea,Gluconacetobacter diazotrophicus, Azospirillum, Sphaerochaeta globus,Flavobacterium columnare, Fluviicola taffensis, Bacteroides coprophilus,Mycoplasma mobile, Lactobacillus farciminis, Streptococcus pasteurianus,Lactobacillus johnsonii, Staphylococcus pseudintermedius, Filifactoralocis, Legionella pneumophila, Suterella wadsworthensis, orCorynebacter diphtheria.

In some embodiments, the Cas9 is a Type II CRISPR system derived from S.pyogenes and the PAM sequence is NGG located on the immediate 3′ end ofthe target specific guide sequence. The PAM sequences of Type II CRISPRsystems from exemplary bacterial species can also include: Streptococcuspyogenes (NGG), Staph aureus (NNGRRT), Neisseria meningitidis (NNNNGATT), Streptococcus thermophilus (NNAGAA) and Treponema denticola(NAAAAC) which are all usable without deviating from the presentinvention.

In one exemplary embodiment, Cas9 sequence can be obtained, for example,from the pX330 plasmid (available from Addgene), re-amplified by PCRthen cloned into pET30 (from EMD biosciences) to express in bacteria andpurify the recombinant 6His tagged protein.

A “Cas9-gNA complex” refers to a complex comprising a Cas9 protein and aguide NA. A Cas9 protein may be at least 60% identical (e.g., at least70%, at least 80%, or 90% identical, at least 95% identical or at least98% identical or at least 99% identical) to a wild type Cas9 protein,e.g., to the Streptococcus pyogenes Cas9 protein. The Cas9 protein mayhave all the functions of a wild type Cas9 protein, or only one or someof the functions, including binding activity, nuclease activity, andnuclease activity.

The term “Cas9-associated guide NA” refers to a guide NA as describedabove. The Cas9-associated guide NA may exist isolated, or as part of aCas9-gNA complex.

Non-CRISPR/Cas System Nucleic Acid-Guided Nucleases

In some embodiments, non-CRISPR/Cas system proteins are used in theembodiments provided herein.

In some embodiments, the non-CRISPR/Cas system proteins can be from anybacterial or archaeal species.

In some embodiments, the non-CRISPR/Cas system protein is isolated,recombinantly produced, or synthetic.

In some embodiments, the non-CRISPR/Cas system proteins are from, or arederived from Aquifex aeolicus, Thermus thermophilus, Streptococcuspyogenes, Staphylococcus aureus, Neisseria meningitidis, Streptococcusthermophiles, Treponema denticola, Francisella tularensis, Pasteurellamultocida, Campylobacter jejuni, Campylobacter lari, Mycoplasmagallisepticum, Nitratifractor salsuginis, Parvibaculum lavamentivorans,Roseburia intestinalis, Neisseria cinerea, Gluconacetobacterdiazotrophicus, Azospirillum, Sphaerochaeta globus, Flavobacteriumcolumnare, Fluviicola taffensis, Bacteroides coprophilus, Mycoplasmamobile, Lactobacillus farciminis, Streptococcus pasteurianus,Lactobacillus johnsonii, Staphylococcus pseudintermedius, Filifactoralocis, Legionella pneumophila, Suterella wadsworthensis,Natronobacterium gregoryi, or Corynebacter diphtheria.

In some embodiments, the non-CRISPR/Cas system proteins can be naturallyoccurring or engineered versions.

In some embodiments, a naturally occurring non-CRISPR/Cas system proteinis NgAgo (Argonaute from Natronobacterium gregoryi).

A “non-CRISPR/Cas system protein-gNA complex” refers to a complexcomprising a non-CRISPR/Cas system protein and a guide NA (e.g. a gRNAor a gDNA). Where the gNA is a gRNA, the gRNA may be composed of twomolecules, i.e., one RNA (“crRNA”) which hybridizes to a target andprovides sequence specificity, and one RNA, the “tracrRNA”, which iscapable of hybridizing to the crRNA. Alternatively, the guide RNA may bea single molecule (i.e., a gRNA) that contains crRNA and tracrRNAsequences.

A non-CRISPR/Cas system protein may be at least 60% identical (e.g., atleast 70%, at least 80%, or 90% identical, at least 95% identical or atleast 98% identical or at least 99% identical) to a wild typenon-CRISPR/Cas system protein. The non-CRISPR/Cas system protein mayhave all the functions of a wild type non-CRISPR/Cas system protein, oronly one or some of the functions, including binding activity, nucleaseactivity, and nuclease activity.

The term “non-CRISPR/Cas system protein-associated guide NA” refers to aguide NA. The non-CRISPR/Cas system protein-associated guide NA mayexist as isolated NA, or as part of a non-CRISPR/Cas system protein-gNAcomplex.

Catalytically Dead Nucleic Acid-Guided Nucleases

In some embodiments, engineered examples of nucleic acid-guidednucleases include catalytically dead nucleic acid-guided nucleases(CRISPR/Cas system nucleic acid-guided nucleases or non-CRISPR/Cassystem nucleic acid-guided nucleases). The term “catalytically dead”generally refers to a nucleic acid-guided nuclease that has inactivatednucleases, for example inactivated HNH and RuvC nucleases. Such aprotein can bind to a target site in any nucleic acid (where the targetsite is determined by the guide NA), but the protein is unable to cleaveor nick the nucleic acid.

Accordingly, the catalytically dead nucleic acid-guided nuclease allowsseparation of the mixture into unbound nucleic acids and catalyticallydead nucleic acid-guided nuclease-bound fragments. Use of a dead nucleicacid-guided nuclease is depicted, for example, in FIG. 9 and FIG. 10, inProtocols 3 and 4, respectively. In one exemplary embodiment, adCas9/gRNA complex binds to the targets determined by the gRNA sequence.The dCas9 bound can prevent cutting by Cas9 while other manipulationsproceed, as pictured in FIG. 10.

In another embodiment, the catalytically dead nucleic acid-guidednuclease can be fused to another enzyme, such as a transposase, totarget that enzyme's activity to a specific site.

In some embodiments, the catalytically dead nucleic acid-guided nucleaseis dCas9, dCpf1, dCas3, dCas8a-c, dCas10, dCse1, dCsy1, dCsn2, dCas4,dCsm2, dCm5, dCsf1, dC2C2, or dNgAgo.

In one exemplary embodiment the catalytically dead nucleic acid-guidednuclease protein is a dCas9.

Nucleic Acid-Guided Nuclease Nickases

In some embodiments, engineered examples of nucleic acid-guidednucleases include nucleic acid-guided nuclease nickases (referred tointerchangeably as nickase nucleic acid-guided nucleases).

In some embodiments, engineered examples of nucleic acid-guidednucleases include CRISPR/Cas system nickases or non-CRISPR/Cas systemnickases, containing a single inactive catalytic domain.

In some embodiments, the nucleic acid-guided nuclease nickase is a Cas9nickase, Cpf1 nickase, Cas3 nickase, Cas8a-c nickase, Cas10 nickase,Cse1 nickase, Csy1 nickase, Csn2 nickase, Cas4 nickase, Csm2 nickase,Cm5 nickase, Csf1 nickase, C2C2 nickase, or a NgAgo nickase.

In one embodiment, the nucleic acid-guided nuclease nickase is a Cas9nickase.

In some embodiments, a nucleic acid-guided nuclease nickase can be usedto bind to target sequence. With only one active nuclease domain, thenucleic acid-guided nuclease nickase cuts only one strand of a targetDNA, creating a single-strand break or “nick”. Depending on which mutantis used, the guide NA-hybridized strand or the non-hybridized strand maybe cleaved. nucleic acid-guided nuclease nickases bound to 2 gNAs thattarget opposite strands can create a double-strand break in the nucleicacid. This “dual nickase” strategy increases the specificity of cuttingbecause it requires that both nucleic acid-guided nuclease/gNA complexesbe specifically bound at a site before a double-strand break is formed.

In exemplary embodiments, a Cas9 nickase can be used to bind to targetsequence. The term “Cas9 nickase” refers to a modified version of theCas9 protein, containing a single inactive catalytic domain, i.e.,either the RuvC- or the HNH-domain. With only one active nucleasedomain, the Cas9 nickase cuts only one strand of the target DNA,creating a single-strand break or “nick”. Depending on which mutant isused, the guide RNA-hybridized strand or the non-hybridized strand maybe cleaved. Cas9 nickases bound to 2 gRNAs that target opposite strandswill create a double-strand break in the DNA. This “dual nickase”strategy can increase the specificity of cutting because it requiresthat both Cas9/gRNA complexes be specifically bound at a site before adouble-strand break is formed.

Capture of DNA can be carried out using a nucleic acid-guided nucleasenickase. This is illustrated in FIG. 6 and FIG. 11, Protocols 2 and 5,respectively. In one exemplary embodiment, as pictured in FIGS. 6 and11, a nucleic acid-guided nuclease nickase cuts a single strand ofdouble stranded nucleic acid, wherein the double stranded regioncomprises methylated nucleotides.

Dissociable and Thermostable Nucleic Acid-Guided Nucleases

In some embodiments thermostable nucleic acid-guided nucleases are usedin the methods provided herein (thermostable CRISPR/Cas system nucleicacid-guided nucleases or thermostable non-CRISPR/Cas system nucleicacid-guided nucleases). In such embodiments, the reaction temperature iselevated, inducing dissociation of the protein; the reaction temperatureis lowered, allowing for the generation of additional cleaved targetsequences. In some embodiments, thermostable nucleic acid-guidednucleases maintain at least 50% activity, at least 55% activity, atleast 60% activity, at least 65% activity, at least 70% activity, atleast 75% activity, at least 80% activity, at least 85% activity, atleast 90% activity, at least 95% activity, at least 96% activity, atleast 97% activity, at least 98% activity, at least 99% activity, or100% activity, when maintained for at least 75° C. for at least 1minute. In some embodiments, thermostable nucleic acid-guided nucleasesmaintain at least 50% activity, when maintained for at least 1 minute atleast at 75° C., at least at 80° C., at least at 85° C., at least at 90°C., at least at 91° C., at least at 92° C., at least at 93° C., at leastat 94° C., at least at 95° C., 96° C., at least at 97° C., at least at98° C., at least at 99° C., or at least at 100° C. In some embodiments,thermostable nucleic acid-guided nucleases maintain at least 50%activity, when maintained at least at 75° C. for at least 1 minute, 2minutes, 3 minutes, 4 minutes, or 5 minutes. In some embodiments, athermostable nucleic acid-guided nucleases maintains at least 50%activity when the temperature is elevated, lowered to 25° C.-50° C. Insome embodiments, the temperature is lowered to 25° C., to 30° C., to35° C., to 40° C., to 45° C., or to 50° C. In one exemplary embodiment,a thermostable enzyme retains at least 90% activity after 1 min at 95°C.

In some embodiments, the thermostable nucleic acid-guided nuclease isthermostable Cas9, thermostable Cpf1, thermostable Cas3, thermostableCas8a-c, thermostable Cas10, thermostable Cse1, thermostable Csy1,thermostable Csn2, thermostable Cas4, thermostable Csm2, thermostableCm5, thermostable Csf1, thermostable C2C2, or thermostable NgAgo.

In some embodiments the thermostable CRISPR/Cas system protein isthermostable Cas9.

Thermostable nucleic acid-guided nucleases can be isolated, for example,identified by sequence homology in the genome of thermophilic bacteriaStreptococcus thermophilus and Pyrococcus furiosus. Nucleic acid-guidednuclease genes can then be cloned into an expression vector. In oneexemplary embodiment, a thermostable Cas9 protein is isolated.

In another embodiment, a thermostable nucleic acid-guided nuclease canbe obtained by in vitro evolution of a non-thermostable nucleicacid-guided nuclease. The sequence of a nucleic acid-guided nuclease canbe mutagenized to improve its thermostability.

Exemplary Compositions of the Invention

In one embodiment, provided herein is a composition comprising a nucleicacid fragment, a nickase nucleic acid-guided nuclease-gNA complex, andlabeled nucleotides. In one exemplary embodiment, provided herein is acomposition comprising a nucleic acid fragment, a nickase Cas9-gRNAcomplex, and labeled nucleotides. In such embodiments, the nucleic acidmay comprise DNA. The nucleotides can be labeled, for example withbiotin. The nucleotides can be part of an antibody-conjugate pair.

In one embodiment, provided herein is a composition comprising a nucleicacid fragment and a catalytically dead nucleic acid-guided nuclease-gNAcomplex, wherein the catalytically dead nucleic acid-guided nuclease isfused to a transposase. In one exemplary embodiment, provided herein isa composition comprising a DNA fragment and a dCas9-gRNA complex,wherein the dCas9 is fused to a transposase.

In one embodiment, provided herein is a composition comprising a nucleicacid fragment comprising methylated nucleotides, a nickase nucleicacid-guided nuclease-gNA complex, and unmethylated nucleotides. In anexemplary embodiment, provided herein is a composition comprising a DNAfragment comprising methylated nucleotides, a nickase Cas9-gRNA complex,and unmethylated nucleotides.

In one embodiment, provided herein is a gDNA complexed with a nucleicacid-guided-DNA endonuclease. In an exemplary embodiment, the nucleicacid-guided-DNA endonuclease is NgAgo.

In one embodiment, provided herein is a gDNA complexed with a nucleicacid-guided-RNA endonuclease.

In one embodiment, provided herein is a gRNA complexed with a nucleicacid-guided-DNA endonuclease.

In one embodiment, provided herein is a gRNA complexed with a nucleicacid-guided-RNA endonuclease. In one embodiment the nucleicacid-guided-RNA endonuclease comprises C2c2.

Kits and Articles of Manufacture

The present application provides kits comprising any one or more of thecompositions described herein, including, but not limited to, adapters,gNAs, gDNAs, gRNAs, gNA libraries, gRNA libraries, gDNA libraries, anucleic acid-guided nuclease, a catalytically dead nucleic acid-guidednuclease, a nickase nucleic acid-guided nuclease, a CRISPR/Cas systemprotein, a nickase CRISPR/Cas system protein, a catalytically deadCRISPR/Cas system protein, Cas9, dCas9, Cas9 nickase, methylatednucleotides, labeled nucleotides, biotinylated nucleotides, avidin,streptavidin, an enzyme capable of initiating nucleic acid synthesis ata nicked site, DNA Polymerase I, TAQ polymerase, bst DNA Polymerase, anenzyme capable of cleaving methylated nucleotides, a Dpn1 enzyme, and anenzyme capable of methylating DNA, for example a Dam/Dcm1methyltransferase).

In one embodiment, the kit comprises a collection or library of gNAswherein the gNAs are targeted to human genomic DNA sequences, forexample particular genes of interest (e.g. cancer genes) SNPs, STRs. Inanother exemplary embodiment, the kit comprises a collection or libraryof gNAs wherein the gNAs are targeted to non-human mammalian DNAsequences. In another exemplary embodiment, the kit comprises acollection or library of gNAs wherein the gNAs are targeted to humanribosomal RNA sequences. In another exemplary embodiment, the kitcomprises a collection or library of gNAs wherein the gNAs are targetedto human mitochondrial DNA sequences.

In one exemplary embodiment, the kit comprises a collection or libraryof gRNAs wherein the gRNAs are targeted to human genomic DNA sequences,for example particular genes of interest (e.g. cancer genes) SNPs, STRs.In another exemplary embodiment, the kit comprises a collection orlibrary of gRNAs wherein the gRNAs are targeted to non-human mammalianDNA sequences. In another exemplary embodiment, the kit comprises acollection or library of gRNAs wherein the gRNAs are targeted to humanribosomal RNA sequences. In another exemplary embodiment, the kitcomprises a collection or library of gRNAs wherein the gRNAs aretargeted to human mitochondrial DNA sequences.

The present application also provides articles of manufacture comprisingany one of the kits described herein. Examples of an article ofmanufacture include vials (including sealed vials).

The following examples are included for illustrative purposes and arenot intend to limit the scope of the invention.

EXAMPLES Example 1: Capture of Mitochondrial DNA from Total HumanGenomic DNA (Protocol 1 for Capture of DNA) Overview

The objective of this method was to capture mitochondrial DNA from alibrary of human genomic DNA, as depicted in FIG. 1. A human tissuespecimen was subjected to DNA extraction protocols, resulting in a DNAsample comprising >99% human DNA and <1% target sequence. The DNA samplewas subjected to sequencing library construction protocols, resulting ina nucleic acid library comprising sequencing indexed adapters. Toproduce a library of target-specific guide RNAs (gRNAs), a library oftarget specific gRNA precursors, each comprising a T7 RNA polymerasepromoter, a human-specific 20-base pair region, and a stem-loop bindingsite for Cas9, was subjected to in vitro transcription, yielding alibrary of target-specific guide RNAs. The library of target-specificguide RNAs was then combined with Cas9 proteins to yield a library ofCas9-gRNA complexes. The Cas9-gRNA complexes were then combined with thenucleic acid library such that the Cas9 cleaved matching target DNAsequences and left other DNA uncleaved. Second adapters were added andallowed to ligate specifically to the 5′ phosphorylated blunt ends ofcleaved DNA. PCR was then used to amplify specifically using the firstand second adapters.

Mitochondrial DNA makes up approximately 0.1-0.2% of total human genomicDNA. To test the precise site-specific cutting of DNA with Cas9,followed by ligation of adapters, referring to FIG. 2, a test DNA (e.g.,a plasmid) 201 was cut with Cas9 at a first location 202 or a secondlocation 203, yielding either first products 204 or second products 205(see, e.g., FIG. 2). Adapters were ligated 206 into the newly availableblunt DNA ends. PCR amplification was performed for AUK-F/P7 orMB1OriR/P7, yielding two separate products per reaction. If the firstlocation was cut, the products were 212 and 1.9 k in size; if the secondlocation was cut, the products were 359 and 1.75 k in size. Verificationwas performed by sequencing.

Results showed that Cas9 cutting followed by ligation of adaptersallowed for specific amplification of target DNA (see, e.g., FIG. 3);sequencing of the amplified DNA showed that ligation of adaptersoccurred only at the location specified by the guide RNA (see, e.g.,FIG. 4).

Mitochondrial DNA was then enriched from a mixture containingpredominantly human nuclear DNA. 25 guide RNAs specific formitochondrial DNA were used, then cut with Cas9 and ligated withadapters, followed by amplification. As seen from FIG. 5, two separatereactions allowed for amplification of mitochondrial DNA whereas areaction without guide RNAs did not amplify any DNA, thus showing thatthe method efficiently amplifies DNA that is under-represented in anygiven library.

Preparation of DNA Libraries

Human genomic DNA libraries were generated by end repairing 500 ng offragmented human genomic DNA (ds DNA fragmentase, NEB, treated for 1hour at 37° C.) using the blunt end repair kit (NEB) for 20 minutes at25° C. Reactions were then heat inactivated at 75° C. for 20 minutes,cooled to 25° C. then ligated to 15 pmoles of P5/Myc adapters for twohours at 25° C. using T4 DNA ligase (NEB). Adapter dimmers were removedusing the NGS cleanup kit (Life Technologies).

Expression of Cas9

Cas9 (from S. pyogenes) was cloned into the pET30 expression vector (EMDbiosciences) to insert the hexahistidine tag immediately upstream of theCas9 start codon. The resulting plasmid was transformed into the Rosetta(DE3) BL21 bacterial strain (EMD biosciences) and grown in 1 L of LBmedia with vigorous aeration until optical density of the culture (OD at600 nm) reached 0.4. The temperature was lowered to 25° C., 0.2 mM IPTGwas added and the culture grown for another four hours. Cells were thenharvested by centrifugation (1,000×g for 20 min at 4° C.), resuspendedin 10 ml binding buffer (20 mM Tris pH8, 0.5 M NaCl, 5 mM Imidazole,0.05% NP40) and lysed by sonication (7×10 second bursts at 30% power,Sonifier 250, Branson). Insoluble cell debris were removed bycentrifugation at 10,000×g for 20 min; supernatant containing solubleprotein was then mixed with 0.4 ml of NTA beads (Qiagen) and loaded ontoa column. Beads were washed three times with 4 ml binding buffer, theneluted with 3×0.5 ml of binding buffer supplemented with 250 mMImidazole. Eluted fractions were then concentrated and buffer exchangedwith storage buffer (10 mM Tris pH8, 0.3 M NaCl, 0.1 mM EDTA, 1 mM DTT,50% glycerol) using a 30,000 MWCO protein concentrator (LifeTechnologies), verified by SDS PAGE followed by Colloidal Blue staining(Life Technologies), quantified, then stored at −20° C. for later use.

A mutant Cas9 nickase, a D10A mutant of S. pyogenes Cas9, can beproduced and purified using the same procedures used to produce Cas9 asabove.

Preparation of gRNA1 and gRNA2

Three oligonucleotides T7-guideRNA1 and 2 (sequences, in 5′ to 3′direction GCCTCGAGCTAATACGACTCACTATAGGGATTTATACAGCACTTTAA (SEQ ID NO:5), and GCCTCGAGCTAATACGACTCACTATAGGGTCTTTTTGGTCCTCGAAG (SEQ ID NO: 6))and stlgR (sequence, GT TTT AGA GCT AGA AAT AGC AAG TTA AAA TAA GGC TAGTCC GTT ATC AAC TTG AAA AAG TGG CAC CGA GTC GGT GCT TTT TTT GGA TCC GATGC (SEQ ID NO: 7)) were ordered and synthesized (IDT). The stlgRoligonucleotide (300 pmol) was sequentially 5′ phosphorylated using T4PNK (New England Biolabs) and then 5′ adenylated sing the 5′adenylationkit (New England Biolabs), according to the manufacturer's instructions.T7-guide RNAs oligonucleotides (5 pmol) and the 5′adenylated stlgR (10pmol) were then ligated using thermostable 5′App DNA/RNA ligase (NewEngland Biolabs) at 65 C for one hour. Ligation reactions were heatinactivated at 90° C. for 5 min, then amplified by PCR (using OneTaq,New England Biolabs, 30 cycles of 95° C. 30 secs, 57° C. 20 secs, 72°C., 20 secs) with primers ForT7 (sequence GCC TCG AGC TAA TAC GAC TCA C(SEQ ID NO: 8)) and gRU (sequence AAAAAAAGCACCGACTCGGTG (SEQ ID NO: 9)).PCR products were purified using PCR cleanup kit (Life Technologies) andverified by agarose gel electrophoresis and sequencing. Verifiedproducts were then used as templates for in vitro transcription.

Preparation of Guide RNA Libraries

T7-guideRNA oligonucleotides (Table 1) and a separate oligonucleotide,stlgR (sequence, GT TTT AGA GCT AGA AAT AGC AAG TTA AAA TAA GGC TAG TCCGTT ATC AAC TTG AAA AAG TGG CAC CGA GTC GGT GCT TTT TTT GGA TCC GAT GC(SEQ ID NO: 7)) were ordered and synthesized (IDT).

The stlgR oligonucleotide (300 pmol) was sequentially 5′ phosphorylatedusing T4 PNK (New England Biolabs) and then 5′ adenylated sing the5′adenylation kit (New England Biolabs), according to the manufacturer'sinstructions. T7-guide RNAs oligonucleotides (5 pmol) and the5′adenylated stlgR (10 pmol) were then ligated using thermostable 5′AppDNA/RNA ligase (New England Biolabs) at 65 C for one hour. Ligationreactions were heat inactivated at 90° C. for 5 min, then amplified byPCR (using OneTaq, New England Biolabs, 30 cycles of 95° C. 30 secs, 57°C. 20 secs, 72° C., 20 secs) with primers ForT7 (sequence GCC TCG AGCTAA TAC GAC TCA C (SEQ ID NO: 8)) and gRU (sequenceAAAAAAAGCACCGACTCGGTG (SEQ ID NO: 9)). PCR products were purified usingPCR cleanup kit (Life Technologies) and verified by agarose gelelectrophoresis and sequencing. Verified products were then used astemplates for in vitro transcription.

TABLE 1 Mito/T7 primers for ligation reactions Series 1GCC TCG AGC TAA TAC GAC TCA CTA TAG GCT TGG ATT AGC GTT TAGT7-1-F (SEQ ID AA NO: 10) T7-13-F (SEQ IDGCC TCG AGC TAA TAC GAC TCA CTA TAG GCT CTT AAA ACT AGG CGG NO: 11) CTAT7-39-F (SEQ IDGCC TCG AGC TAA TAC GAC TCA CTA TAG ATT TAC ACT CAC AAC ACC NO: 12) CTT7-41-F (SEQ IDGCC TCG AGC TAA TAC GAC TCA CTA TAG AAC AGC TAT CCA TTG GTC NO: 13) TTT7-43-F (SEQ IDGCC TCG AGC TAA TAC GAC TCA CTA TAG GCA GCC GGA AGC CTA TTC NO: 14) GCT7-61-F (SEQ IDGCC TCG AGC TAA TAC GAC TCA CTA TAG GTA ATG AGG ATG TAA GCC NO: 15) CGT7-63-F (SEQ IDGCC TCG AGC TAA TAC GAC TCA CTA TAG ATA TTT ACA AGA GGA AAA NO: 16) CCT7-65-F (SEQ IDGCC TCG AGC TAA TAC GAC TCA CTA TAG GTT TGA AGC TTA GGG AGA NO: 17) GCTT7-67-F (SEQ IDGCC TCG AGC TAA TAC GAC TCA CTA TAG GTA TGG CTT TGA AGA AGG NO: 18) CGSeries 2 GCC TCG AGC TAA TAC GAC TCA CTA TAG TAG ATG ACG GGT TGG GCCT7mtgRNA3 AG (SEQ ID NO: 19) T7mtgRNA7GCC TCG AGC TAA TAC GAC TCA CTA TAG AGC TTT ACA GTG GGC TCT(SEQ ID NO: 20) AG T7mtgRNA11GCC TCG AGC TAA TAC GAC TCA CTA TAG ATG GCA GCT TCT GTG GAA(SEQ ID NO: 21) CG T7mtgRNA15GCC TCG AGC TAA TAC GAC TCA CTA TAG GTG GTA AGG GCG ATG AGT(SEQ ID NO: 22) GT T7mtgRNA31GCC TCG AGC TAA TAC GAC TCA CTA TAG TCC ATA ACG CTC CTC ATA(SEQ ID NO: 23) CT T7mtgRNA33GCC TCG AGC TAA TAC GAC TCA CTA TAG TCT CCC TTC ACC ATT TCC(SEQ ID NO: 24) GA

In Vitro Transcription

Verified products were then used as templates for in vitro transcriptionreactions using the HiScribe T7 transcription kit (New England Biolabs).500-1000 ng of template was incubated overnight at 37° C. according tothe manufacturer's instruction. To transcribe the guide libraries intoguide RNA, we assembled the following in vitro transcription reactionmixture: 10 μl purified library (˜500 ng), 6.5 μl of H2O, of 2.25 μl ofATP, 2.25 μl of CTP, 2.25 μl of GTP, 2.25 μl of UTP, 2.25 μl 10×reaction buffer (NEB) and 2.25 μl of T7 RNA polymerase mix. The reactionwas incubated at 37° C. for 24 hours, then purified using the RNAcleanup kit (Life Technologies), eluted into 100 μl of RNase-free water,quantified and stored at −20° C. until use.

DNA-Specific Cas9-Mediated Fragmentation

For cutting the test DNA test guide RNA 1 and 2 were used separately.For cutting and enriching the mitochondrial DNA from a human genomic DNAlibrary, guide RNA series 1 and 2 were used. Diluted guide RNA (1 ul,equivalent to 2 pmol) was combined with 3 ul 10× Cas9 reaction buffer(NEB), 20 ul H2O and 1 ul of recombinant Cas9 enzyme (NEB, 1 pmol/ul). Acontrol reaction using a control guide RNA targeting the followingsequence (5′-GGATTTATACAGCACTTTAA-3′(SEQ ID NO: 25)) was performedseparately, using the same parameters. This sequence is absent fromeither the human chromosomal or mitochondrial DNA. Reactions wereincubated for 15 min at 37° C., then supplemented with 5 μl diluted DNAlibrary (50 pg/μl) and incubation at 37° C. continued for 90 min. Thereactions were terminated by adding RNase A (Thermo Fisher Scientific)at a 1:100 dilution, then purifying the DNA using a PCR cleanup kit(LifeTechnologies) and eluting in 30 ul 10 mM Tris-Cl pH 8. Reactionswere then stored at −20° C. until use.

Ligation of Adapters and PCR Analysis

For the test DNA, reactions after Cas9 digestion were incubated with 15pmoles of P5/P7 adapters and T4 DNA ligase (NEB) for one hour at 25° C.Ligations were then used as templates for PCR using the TestDNA-F primer(sequence ATGCCGCAGCACTTGG (SEQ ID NO: 26)) and P5 primer (sequenceAATGATACGGCGACCACCGA (SEQ ID NO: 27)). Succesful PCR products wereconfirmed by agarose gel electrophoresis sequenced using the TestDNA-Fprimer (ElimBio), to show cutting and ligation occurred at the targetDNA sequence.

Elimination of Adapters Ligated at Old Ends

For the enrichment of mitochondrial DNA, after Cas9 digestion, reactionswere incubated with 15 pmoles of Flag5/P7 adapters and T4 DNA ligase(NEB) for one hour at 25° C. Multiple molecular biology methods can beemployed to eliminate ligation of adapters at the ends of the old P5 andP7 adapters from the original library, for example with enzymetreatment. Reactions were then used as templates for PCR using the P7(sequence CAAGCAGAAGACGGCATACGA (SEQ ID NO: 28)) and P5 primers(sequence AATGATACGGCGACCACCGA (SEQ ID NO: 27)). Successful PCR productswere confirmed by agarose gel electrophoresis.

Example 2: Using Cas9 Nickase to Label, then Purify Test DNA from a DNAMixture (Protocol 2 for Capture of DNA) Overview

The purpose of this method was to capture a region of interest (e.g.,SNP, STR, etc.) from a library of human genomic DNA, as depicted forexample in FIG. 6. To apply this protocol, a test DNA containing a sitefor a nicking enzyme guided by target-specific guide RNAs (NtAlwI (NEB))was mixed at 1% or 5% into another pool of DNA that does not containthis site. Nickase Cas9 cleaves only at target sequence and only cutsone strand of DNA. Nickase was used to nick target sequences. Singlestrand cuts (nicks) are a substrate for DNA polymerase I which can beused to replace the DNA downstream of the nick with biotin labeled DNA.Biotinylated DNA of interest was then purified, amplified, andsequenced.

The mixture of DNA with 701 and without 702 target sites for nicking(e.g., GGATC) 703 was nicked 704 using NtAlwI (see, e.g., FIG. 7). DNAthat does not have nickase sites was present in 100× excess compared toDNA of interest with the nickase sites. Single strand cuts (nicks) are asubstrate for DNA polymerase I which are used to replace 705 the DNAdownstream of the nick with biotin labeled DNA. Biotin-labeled DNA ofinterest was isolated by streptavidin binding and washing 706. PCRamplification was performed with specific primers 707, yieldingamplified regions of interest 708 and amplified unlabeled sequences 709present, for example, due to non-specific labeling or capture.

Two specific PCR reactions (one for the test DNA with regions ofinterest, the other for other DNA without regions of interest) showedthat the test DNA had been enriched approximately 50-fold (see, e.g.,FIG. 8).

Expression and Purification of Cas9 Nickase

A mutant Cas9 nickase, a D10A mutant of S. pyogenes Cas9, can beproduced and purified using the same procedures used to produce Cas9 asabove.

Sequence Specific Cas9 Nickase-Mediated Nicking

Diluted guide RNA, targeting the following sequence5′-GGATTTATACAGCACTTTAA-3′ (SEQ ID NO: 29) (1 ul, equivalent to 2 pmol)was combined with 3 ul 10× Cas9 reaction buffer (NEB), 20 ul H2O and 1ul of recombinant Cas9 Nickase enzyme (10 pmol/ul). This target sequenceis only present on the target DNA (that makes either 5% or 1% of thetotal DNA). Reactions were incubated for 15 min at 37° C., thensupplemented with 5 μl diluted DNA (100 ng total) and incubation at 37°C. continued for 90 min. The reactions were terminated by adding RNase A(Thermo Fisher Scientific) at a 1:100 dilution, then purifying the DNAusing a PCR cleanup kit (LifeTechnologies) and eluting in 30 ul 10 mMTris-Cl pH 8. Reactions were then stored at −20° C. until use.

Biotin Nick Translation

Nicked DNA was incubated with E. coli DNA polymerase I which has 5′>3′exonuclease activity, and is capable of initiating DNA synthesis atnicks, thus allowing it to replace nucleotides downstream of a nick withlabeled nucleotides (in the case of this procedure, biotin-labelednucleotides). Nick labeling reactions were performed in 20 ul of DNApolymerase buffer (NEB) with 1 unit of E. coli DNA polymerase I (NEB)and 0.02 mM each of dCTP, dGTP and dTTP, 0.01 mM of dATP and 0.01 mMbiotin-C14 labeled dATP (LifeTechnologies) for 30 min at 25° C.Reactions were terminated by adding 1 mM EDTA.

Enrichment of Biotin Labeled DNA

Streptavidin C1 beads (5 ul per reaction, LifeTechnologies) wereresuspended in 1 ml binding buffer (50 mM Tris-Cl pH 8, 1 mM EDTA, 0.1%Tween20), bound to a magnetic rack, and washed twice with bindingbuffer. Beads were then resuspended in 30 ul of binding buffer and mixedwith the nick translation reaction, then incubated at 25° C. for 30minutes. Beads were captured using the magnetic rack and washed fourtimes with 0.5 ml binding buffer, then three times with 0.5 ml of 10 mMTris-Cl pH 8. Beads were then resuspended in 20 ul 10 mM Tris-Cl pH 8,then used as templates for PCR to determine the proportion of test DNAand other DNA.

Example 3: Use of a Catalytically Dead Nucleic Acid-GuidedNuclease-Transposase Fusion to Insert Adaptors in Human Genomic LibraryFollowed by Enriching for Specific SNPs (Protocol 3 for Capture of DNA)

In this example, catalytically dead nucleic acid-guidednuclease-transposase fusion protein (e.g. a dCas9-transposase fusionprotein) is expressed and purified from E. coli as described for theCas9 purification. The fusion protein is complexed with adapters(Nextera) then with guide NAs (e.g. gRNAs) targeting the regions ofinterest (e.g. human SNPs). Then the complex is added to human genomicDNA; regions of interest are targeted by the catalytically dead nucleicacid-guided nuclease, bringing the trasnposase-adapter complex in closeproximity to the regions of interest, allowing insertion of theadapters. Human SNPs can then be amplified by PCR and then sequencedusing MiSeq; thus human SNPs can be enriched from human genomic DNA.

Example 4: Using Catalytically Dead Nucleic Acid-Guided Nuclease andNucleic Acid-Guided Nuclease to Protect Mitochondrial DNA and DigestRemaining Human Nuclear DNA from a Human Genomic DNA Library (Protocol 4for Capture of DNA)

A human genomic DNA library with P5/P7 adapters from a clinical,forensic or environmental sample is obtained. To enrich for certainregions (e.g. SNPs) guide RNAs targeting these regions are made andincubated with catalytically dead nucleic acid-guided nuclease (e.g.dCas9) then added to the human genomic DNA library for 20 minutes at 37C. Then, a library of guide NAs covering the human genome complexed withactive nucleic acid-guided nuclease (e.g. Cas9) is added. Thecatalytically dead nucleic acid-guided nuclease will remain bound at thetarget locations and protect the regions of interests (e.g. SNPs) frombeing cleaved and becoming non PCR-amplifiable and sequence-able. Thus,the DNA of interest remains intact while all other DNA will be cleavedand eliminated. DNA of interest is recovered from the reactions usingthe PCR cleanup kit, PCR amplified and sequenced using MiSeq.

For proof of concept testing, mitochondrial DNA is enriched from a totalhuman genomic DNA library. Mitochondrial specific guide RNAs are addedto dCas9, then added to the library. Then, random guide RNAs complexedwith Cas9 degrade any sequence, except for those inaccessible becausealready protected by bound dCas9. This allows enrichment of themitochondrial DNA to levels far higher than the original 0.1-0.2%.

Example 5: Using a Nucleic Acid-Guided Nuclease Nickase (e.g. Cas9Nickase) to Protect and then Enrich SNPs from Human Genomic DNA byReplacing Methylated DNA with Unmethylated DNA (Protocol 5 for Captureof DNA) Overview

The objective of this method is to capture a region of interest from alibrary of human genomic DNA, as depicted in FIG. 11. As proof ofprinciple, a test DNA containing a site for a nicking enzyme, NtBbvCI(NEB) was mixed at 1% or 5% into another pool of DNA that does notcontain this site. The entire mixture was then treated with Dammethyltransferase to add methyl groups to all GATC sequences. Both testDNA and other DNA contain GATC motifs in their sequence. The mixture wasthen nicked using NtBbvCI, then incubated with DNA polymerase I andunlabeled nucleotides (dATP, dCTP, dGTP, dTTP), then heat inactivated at75° C. for 20 minutes. The mixture is then digested with DpnI, whichonly digests methylated DNA; unmethylated or hemimethylated DNA will notbe digested. FIG. 12 shows that methylation of test DNA renders itsusceptible to DpnI mediated cleavage.

Expression and Purification of Cas9 Nickase

A mutant Cas9 nickase, a D10A mutant of S. pyogenes Cas9, can beproduced and purified using the same procedures used to produce Cas9 asabove.

Sequence Specific Cas9 Nickase-Mediated Nicking

Diluted guide RNA, targeting the following sequence5′-GGATTTATACAGCACTTTAA-3′ (SEQ ID NO: 29) (1 ul, equivalent to 2 pmol)is combined with 3 ul 10× Cas9 reaction buffer (NEB), 20 ul H2O and 1 ulof recombinant Cas9 Nickase enzyme (10 pmol/ul). This target sequence isonly present on the target DNA (that makes either 5% or 1% of the totalDNA). Reactions are incubated for 15 min at 37° C., then supplementedwith 5 μl diluted DNA (100 ng total) and incubation at 37° C. continuedfor 90 min. The reactions are terminated by adding RNase A (ThermoFisher Scientific) at a 1:100 dilution, then purifying the DNA using aPCR cleanup kit (LifeTechnologies) and eluting in 30 ul 10 mM Tris-Cl pH8. Reactions are then stored at −20° C. until use.

Unlabeled DNA Nick Translation

Nicked DNA is incubated with E. coli DNA polymerase I which has 5′>3′exonuclease activity, and is capable of initiating DNA synthesis atnicks, thus allowing it to replace nucleotides downstream of a nick withlabeled nucleotides (in the case of this procedure, biotin-labelednucleotides). Nick labeling reactions are performed in 20 ul of DNApolymerase buffer (NEB) with 1 unit of E. coli DNA polymerase I (NEB)and 0.02 mM each of dATP, dCTP, dGTP and dTTP for 30 min at 25° C.Reactions are terminated by heat inactivation at 75° C. for 20 minutes.

Digestion with DpnI

Reactions are then incubated with DpnI (NEB) for one hour at 37° C. DNAis recovered using the PCR cleanup kit (LifeTechnologies) and then usedas template for PCR using test DNA specific primers.

Example 6: Using a Nucleic Acid-Guided Nuclease Nickase (e.g. Cas9Nickase) to Generate Long 3′ Overhangs Flanking Regions of Interests,then Ligating Adapters to these Overhangs to Enrich Target DNA (Protocol6 for Capture of DNA)

The objective of this method can be used to enrich a region of DNA, fromany DNA source (e.g., library, genomic, or PCR), as depicted for examplein FIG. 13. The nucleic acid-guided nuclease nickase (e.g. Cas9Nickase)’ can be targeted to proximal sites using two guide NAs (e.g.gRNAs) and, resulting in nicking of DNA at each location. Alternatively,an adapter can be ligated on only one side, then filled in, then anadapter can be ligated on the other side. The two nicks can be close toeach other (e.g., within 10 to 15 bp). Single nicks may be generated innon-target molecules. Because of the proximity of the two nicking sites,a double stranded break can be created when the reaction is heated, e.g.to 65° C., resulting in long (e.g., 10-15 bp) 3′ overhangs. Theseoverhangs can be recognized by a thermostable single stranded DNA/RNAligase, such as a Thermostable 5′App DNA/RNA ligase to allow forsite-specific ligation of single stranded adapters. The ligase can, forexample, only recognize long 3′ overhangs, thus ensuring that adapterswill not be ligated at other sites. This process can be repeated usingthe nucleic acid-guided nuclease nickase and guide NA targeting on theother side of the region of interest, followed by ligation as aboveusing a second single stranded adapter. Once two adapters have beenligated on either side of the region of interest, the region can beamplified or sequenced directly.

What is claimed is:
 1. A method of capturing target nucleic acidsequences comprising: (a) providing a sample comprising a plurality ofadapter-ligated nucleic acids, wherein the nucleic acids are ligated toa first adapter at one end and ligated to a second adapter at the otherend; (b) contacting the sample with a plurality of nucleic acid-guidednuclease-gNA complexes, wherein the gNAs are complementary to targetedsites of interest contained in a subset of the nucleic acids, therebygenerating a plurality of nucleic acid fragments ligated to a first orsecond adapter at one end and no adapter at the other end; and (c)contacting the plurality of nucleic acid fragments with third adapters,thereby generating a plurality of nucleic acid fragments ligated toeither the first or second adapter at one end and the third adapter atthe other end.
 2. The method of claim 1, wherein the nucleic acid-guidednuclease is a CRISPR/Cas system protein.
 3. The method of claim 1,wherein the nucleic acid-guided nuclease is a non-CRISPR/Cas systemprotein.
 4. The method of claim 1, wherein the nucleic acid-guidednuclease is selected from the group consisting of CAS Class I Type I,CAS Class I Type III, CAS Class I Type IV, CAS Class II Type II, and CASClass II Type V.
 5. The method of claim 1, wherein the nucleicacid-guided nuclease is selected from the group consisting of Cas9,Cpf1, Cas3, Cas8a-c, Cas10, Cse1, Csy1, Csn2, Cas4, Csm2, Cmr5, Csf1,C2c2, and NgAgo.
 6. The method of any one of claims 1 to 5, wherein thegNAs are gRNAs.
 7. The method of any one of claims 1 to 5, wherein thegNAs are gDNAs.
 8. The method of any one of claims 1 to 7, wherein thecontacting with the plurality of nucleic acid-guided nuclease-gNAcomplexes cleaves the targeted sites of interest contained in a subsetof the nucleic acids, thereby generating a plurality of nucleic acidfragments comprising a first or second adapter at one end and no adapterat the other end.
 9. The method of any one of claims 1 to 8, wherein themethod further comprises amplifying the product of step (c) using firstor second and third adapter-specific PCR.
 10. The method of any one ofclaims 1 to 9, wherein the nucleic acids are selected from the groupconsisting of single stranded DNA, double stranded DNA, single strandedRNA, double stranded RNA, and a DNA/RNA hybrid.
 11. The method of claim10, wherein the nucleic acids are double stranded DNA.
 12. The method ofany one of claims 1 to 11, wherein nucleic acids are from genomic DNA.13. The method of claim 12, wherein the genomic DNA is human.
 14. Themethod of any one of claims 1 to 13, wherein the nucleic acids which areadapter-ligated are from 20 bp to 5000 bp in length.
 15. The method ofany one of claims 1 to 14, wherein the targeted sites of interest aresingle nucleotide polymorphisms (SNPs), short tandem repeats (STRs),cancer genes, inserts, deletions, structural variations, exons, geneticmutations, or regulatory regions.
 16. The method of claim 9, whereinamplified products are used for cloning, sequencing, or genotyping. 17.The method of any one of claims 1 to 16, wherein the adapters are from20 bp to 100 bp in length.
 18. The method of any one of claims 1 to 17,wherein the adapters comprise primer binding sites.
 19. The method ofany one of claims 1 to 18, wherein the adapters comprise sequencingadapters or restriction sites.
 20. The method of any one of claims 1 to19, wherein the targeted sites of interest represent less than 50% ofthe total nucleic acid in the sample.
 21. The method of any one ofclaims 1 to 20, wherein the sample is obtained from a biological samplea clinical sample, a forensic sample, or an environmental sample. 22.The method of any one of claims 1 to 21, wherein the first and secondadapters are identical.
 23. The method of any one of claims 1 to 21,wherein the first and second adapters are different.
 24. The method ofany one of claims 1 to 23, wherein the sample comprises a sequencinglibrary.
 25. A method of introducing labeled nucleotides at targetedsites of interest comprising: (a) providing a sample comprising aplurality of nucleic acid fragments; (b) contacting the sample with aplurality of nucleic acid-guided nuclease nickase-gNA complexes whereinthe gNAs are complementary to targeted sites of interest in the nucleicacid fragments, thereby generating a plurality of nicked nucleic acidfragments at the targeted sites of interest; and (c) contacting theplurality of nicked nucleic acid fragments with an enzyme capable ofinitiating nucleic acid synthesis at a nicked site, and labelednucleotides, thereby generating a plurality of nucleic acid fragmentscomprising labeled nucleotides in the targeted sites of interest. 26.The method of claim 25, wherein the nucleic acid-guided nuclease nickaseis selected from the group consisting of CAS Class I Type I nickase, CASClass I Type III nickase, CAS Class I Type IV nickase, CAS Class II TypeII nickase, and CAS Class II Type V nickase.
 27. The method of claim 25,wherein the nucleic acid-guided nuclease nickase is selected from thegroup consisting of Cas9 nickase, Cpf1 nickase, Cas3 nickase, Cas8a-cnickase, Cas10 nickase, Cse1 nickase, Csy1 nickase, Csn2 nickase, Cas4nickase, Csm2 nickase, Cm5 nickase, Csf1 nickase, C2C2 nickase, CPF1nickase, and NgAgo nickase.
 28. The method of any one of claims 25 to27, wherein the gNAs are gRNAs.
 29. The method of any one of claims 25to 27, wherein the gNAs are gDNAs.
 30. The method of any one of claims25 to 29, wherein the nucleic acid fragments are selected from the groupconsisting of single stranded DNA fragments, double stranded DNAfragments, single stranded RNA fragments, double stranded RNA fragments,and a DNA/RNA hybrid fragments.
 31. The method of claim 30, wherein thenucleic acid fragments are double stranded DNA fragments.
 32. The methodof claim 31, wherein double stranded DNA fragments are from genomic DNA.33. The method of claim 32, wherein the genomic DNA is human.
 34. Themethod of any one of claims 25 to 33, wherein the nucleic acid fragmentsare from 20 bp to 5000 bp in length.
 35. The method of any one of claims25 to 34, wherein the targeted sites of interest are single nucleotidepolymorphisms (SNPs), short tandem repeats (STRs), cancer genes,inserts, deletions, structural variations, exons, genetic mutations, orregulatory regions.
 36. The method of any one of claims 25 to 35,wherein the targeted sites of interest represent less than 50% of thetotal nucleic acid in the sample.
 37. The method of any one of claims 25to 36, wherein the sample is obtained from a biological sample aclinical sample, a forensic sample, or an environmental sample.
 38. Themethod of any one of claims 25 to 37, wherein the labeled nucleotidesare biotinylated nucleotides.
 39. The method of any one of claims 25 to37, wherein the labeled nucleotides are part of an antibody conjugatepair.
 40. The method of claim 38, further comprising contacting thenucleic acid fragments comprising biotinylated nucleotides with avidinor strepavidin, thereby capturing the targeted sites of interest. 41.The method of any one of claims 25 to 40, wherein the enzyme capable ofinitiating nucleic acid synthesis at a nicked site is DNA Polymerase I,a Klenow fragment, a TAQ polymerase, or a Bst DNA Polymerase.
 42. Themethod of any one of claims 25 to 41, wherein the nucleic acid-guidednuclease nickase nicks the 5′ end of the nucleic acid fragments.
 43. Themethod of any one of claims 25 to 42, wherein the nucleic acid fragmentsare from 20 bp to 5000 bp in length.
 44. The method of any one of claims25 to 43, wherein the sample is obtained from a biological sample aclinical sample, a forensic sample, or an environmental sample.
 45. Amethod of capturing target nucleic acid sequences of interestcomprising: (a) providing a sample comprising a plurality ofadapter-ligated nucleic acids, wherein the nucleic acids are ligated toa first adapter at one end and are ligated to a second adapter at theother end; and (b) contacting the sample with a plurality ofcatalytically dead nucleic acid-guided nuclease-gNA complexes, whereinthe catalytically dead nucleic acid-guided nuclease is fused to atransposase, wherein the gRNAs are complementary to targeted sites ofinterest contained in a subset of the nucleic acids, and wherein thecomplexes are loaded with a plurality of third adapters, to generate aplurality of nucleic acids fragments comprising either a first or secondadapter at one end, and a third adapter at the other end.
 46. The methodof claim 45, wherein the method further comprises amplifying the productof step (b) using first or second adapter and third adapter-specificPCR.
 47. The method of claim 45, wherein the catalytically dead nucleicacid-guided nuclease is derived from a CRISPR/Cas system protein. 48.The method of claim 45, wherein the catalytically dead nucleicacid-guided nuclease is derived from a non-CRISPR/Cas system protein.49. The method of claim 45, wherein the catalytically dead nucleicacid-guided nuclease is selected from the group consisting of dead CASClass I Type I, dead CAS Class I Type III, dead CAS Class I Type IV,dead CAS Class II Type II, and dead CAS Class II Type V.
 50. The methodof claim 45, wherein the catalytically dead nucleic acid-guided nucleaseis selected from the group consisting of dCas9, dCpf1, dCas3, dCas8a-c,dCas10, dCse1, dCsy1, dCsn2, dCas4, dCsm2, dCmr5, dCsf1, dC2C2, anddNgAgo.
 51. The method of any one of claims 45 to 50, wherein the gNAsare gRNAs.
 52. The method of any one of claims 45 to 50, wherein thegNAs are gDNAs.
 53. The method of any one of claims 45 to 52, whereinnucleic acids sequences are from genomic DNA.
 54. The method of claim53, wherein the genomic DNA is human.
 55. The method of any one ofclaims 45 to 54, wherein the catalytically dead nucleic acid-guidednuclease is fused to the N-terminus of the transposase.
 56. The methodof any one of claims 45 to 54, wherein the catalytically dead nucleicacid-guided nuclease is fused to the C-terminus of the transposase. 57.The method of any one of claims 45 to 56, wherein the nucleic acids,which are adapter ligated, are from 20 bp to 5000 bp in length.
 58. Themethod of any one of claims 45 to 57, wherein the contacting of step (b)allows for the insertion of the second adapter into the targeted nucleicacid sequences.
 59. The method of any one of claims 45 to 58, whereinthe targeted sites of interest are single nucleotide polymorphisms(SNPs), short tandem repeats (STRs), cancer genes, inserts, deletions,structural variations, exons, genetic mutations, or regulatory regions.60. The method of claim 46, wherein amplified products are used forcloning, sequencing, or genotyping.
 61. The method of any one of claims45 to 59, wherein the adapters are from 20 bp to 100 bp in length. 62.The method of any one of claims 45 to 61, wherein the adapters compriseprimer binding sites.
 63. The method of any one of claims 45 to 62,wherein the adapters comprise sequencing adapters or restriction sites.64. The method of any one of claims 45 to 63, wherein the targeted sitesof interest represent less than 50% of the total nucleic acid in thesample.
 65. The method of any one of claims 45 to 64, wherein the sampleis obtained from a biological sample a clinical sample, a forensicsample, or an environmental sample.
 66. A method of capturing targetnucleic acid sequences of interest comprising: (a) providing a samplecomprising a plurality of adapter-ligated nucleic acids, wherein thenucleic acids are ligated to the adapter at the 5′ end and 3′ ends; (b)contacting the sample with a plurality of catalytically dead nucleicacid-guided nuclease-gNA complexes, wherein the gNAs are complementaryto targeted sites of interest contained in a subset of the nucleicacids, thereby generating a plurality of nucleic acids adapter-ligatedat the 5′ and 3′ ends, bound to a catalytically dead nucleic acid-guidednuclease-gNA complex; and (c) contacting the sample with a plurality ofcatalytically dead nucleic acid-guided nuclease-gNA complexes, whereinthe gNAs are complementary to both targeted sites of interest andtargeted sites not of interest in the nucleic acids, thereby generatinga plurality of nucleic acid fragments comprising nucleic acid sequencesnot of interest, adapter ligated at only one of the 5′ or 3′ ends. 67.The method of claim 66, wherein the catalytically dead nucleicacid-guided nuclease is a CRISPR/Cas system protein.
 68. The method ofclaim 66, wherein the catalytically dead nucleic acid-guided nuclease isa non-CRISPR/Cas system protein.
 69. The method of claim 66, wherein thecatalytically-dead nucleic acid-guided nuclease is selected from thegroup consisting of dead CAS Class I Type I, dead CAS Class I Type III,dead CAS Class I Type IV, dead CAS Class II Type II, and dead CAS ClassII Type V.
 70. The method of claim 66, wherein the catalytically deadnucleic acid-guided nuclease is selected from the group consisting ofdCas9, dCpf1, dCas3, dCas8a-c, dCas10, dCse1, dCsy1, dCsn2, dCas4,dCsm2, dCm5, dCsf1, dC2C2, dCPF1, and dNgAgo.
 71. The method of any oneof claims 66 to 70, wherein the gNAs are gRNAs.
 72. The method of anyone of claims 66 to 70, wherein the gNAs are gDNAs.
 73. The method ofany one of claims 66 to 72, wherein the contacting of step (c) does notdisplace the plurality of nucleic acids adapter-ligated at the 5′ and 3′ends, bound to a catalytically dead nucleic acid-guided nuclease-gNAcomplex of step (b).
 74. The method of any one of claims 66 to 73,wherein the contacting in step (d) cleaves the targeted sites not ofinterest contained in a subset of the nucleic acids, thereby generatinga plurality of nucleic acid fragments comprising nucleic acid sequencesnot of interest, adapter ligated at only one of the 5′ or 3′ ends. 75.The method of any one of claims 66 to 74, wherein the method furthercomprises removing the bound catalytically dead nucleic acid-guidednuclease-gNA complex and amplifying the product of step (b) usingadapter-specific PCR.
 76. The method of any one of claims 66 to 75,wherein the nucleic acids are selected from the group consisting ofsingle stranded DNA, double stranded DNA, single stranded RNA, doublestranded RNA, and a DNA/RNA hybrid.
 77. The method of claim 76, whereinthe nucleic acids are double stranded DNA.
 78. The method of any one ofclaims 66 to 77, wherein nucleic acids are from genomic DNA.
 79. Themethod of claim 78, wherein the genomic DNA is human.
 80. The method ofany one of claims 66 to 79, wherein the nucleic acids adapter-ligated atthe 5′ends and the 3′ends are from 20 bp to 5000 bp.
 81. The method ofany one of claims 66 to 80, wherein the targeted sites of interest aresingle nucleotide polymorphisms (SNPs), short tandem repeats (STRs),cancer genes, inserts, deletions, structural variations, exons, geneticmutations, or regulatory regions.
 82. The method of claim 75, whereinamplified products are used for cloning, sequencing, or genotyping. 83.The method of any one of claims 66 to 82, wherein the adapters are from20 bp to 100 bp in length.
 84. The method of any one of claims 66 to 82,wherein the adapters comprise primer binding sites.
 85. The method ofany one of claims 66 to 82, wherein the adapters comprise sequencingadapters or restriction sites.
 86. The method of any one of claims 66 to82, wherein the targeted sites of interest represent less than 50% ofthe total nucleic acid in the sample.
 87. The method of any one ofclaims 66 to 82, wherein the sample is obtained from a biological samplea clinical sample, a forensic sample, or an environmental sample.
 88. Amethod of capturing target nucleic acid sequences of interestcomprising: (a) providing a sample comprising a plurality of nucleicacid sequences, wherein the nucleic acid sequences comprise methylatednucleotides, and wherein the nucleic acid sequences are adapter ligatedon the 5′ and 3′ ends; (b) contacting the sample with a plurality ofnucleic acid-guided nuclease nickase-gNA complexes, wherein the gNAs arecomplementary to targeted sites of interest in a subset of the nucleicacid sequences, thereby generating a plurality of nicked sites ofinterest in the subset of the nucleic acid sequences, and wherein thetarget nucleic acid sequences are adapter ligated on the 5′ and 3′ ends;(c) contacting the sample with an enzyme capable of initiating DNAsynthesis at a nicked site, and unmethylated nucleotides, therebygenerating a plurality of nucleic acid sequences comprising unmethylatednucleotides in the targeted sites of interest and wherein the nucleicacid sequences are adapter ligated on the 5′ and 3′ ends; and (d)contacting the sample with an enzyme capable of cutting methylatednucleic acids, thereby generating a plurality of nucleic acid fragmentscomprising methylated nucleic acids, wherein the plurality of nucleicacid fragments comprising methylated nucleic acids that are adapterligated on at most one of the 5′ and 3′ ends.
 89. The method of claim88, wherein the nucleic acid-guided nuclease nickase is selected fromthe group consisting of CAS Class I Type I nickase, CAS Class I Type IIInickase, CAS Class I Type IV nickase, CAS Class II Type II nickase, andCAS Class II Type V nickase.
 90. The method of claim 88, wherein thenucleic acid-guided nuclease nickase is selected from the groupconsisting of Cas9 nickase, Cpf1 nickase, Cas3 nickase, Cas8a-c nickase,Cas10 nickase, Cse1 nickase, Csy1 nickase, Csn2 nickase, Cas4 nickase,Csm2 nickase, Cmr5 nickase, Csf1 nickase, C2C2 nickase, and NgAgonickase.
 91. The method of claim 88, wherein the gNAs are gRNAs.
 92. Themethod of claim 88, wherein the gNAs are gDNAs.
 93. The method of anyone of claims 88 to 92, wherein the DNA is double stranded DNA.
 94. Themethod of claim 93, wherein the double stranded DNA is from genomic DNA.95. The method of claim 94, wherein the genomic DNA is human.
 96. Themethod of any one of claims 88 to 95, wherein the DNA sequences are from20 bp to 5000 bp in length.
 97. The method of any one of claims 88 to96, wherein the targeted sites of interest are single nucleotidepolymorphisms (SNPs), short tandem repeats (STRs), cancer genes,inserts, deletions, structural variations, exons, genetic mutations, orregulatory regions.
 98. The method of any one of claims 88 to 97,wherein the targeted sites of interest represent less than 50% of thetotal DNA in the sample.
 99. The method of any one of claims 88 to 98,wherein the sample is obtained from a biological sample a clinicalsample, a forensic sample, or an environmental sample.
 100. The methodof any one of claims 88 to 99, wherein the enzyme capable of initiatingnucleic acid synthesis at a nicked site is a DNA Polymerase I, Klenowfragment a TAQ polymerase, or a Bst DNA Polymerase.
 101. The method ofany one of claims 88 to 100, wherein the nucleic acid-guided nucleasenickase nicks the 5′ end of the DNA sequences.
 102. The method of anyone of claims 88 to 101, wherein the enzyme capable of cuttingmethylated DNA is DpnI.
 103. A method of capturing target DNA sequencesof interest comprising: (a) contacting the sample with a plurality ofnucleic acid-guided nuclease nickase-gNA complexes, wherein the gNAs arecomplementary to targeted sites of interest flanking a region ofinterest in a subset of the DNA sequences, thereby generating aplurality of nicked DNA at sites adjacent to the regions of interest;(b) heating the sample to 65° C. thereby causing cause nicks in closeproximity to generate a double stranded breaks; (c) contacting thedouble stranded breaks with a thermostable ligase thereby allowingligation of adapter sequences at these sites only; and (d) repeatingsteps a-c to place a second adapter on the other side of the region ofinterest, thus allowing enrichment of the region of interest.
 104. Themethod of claim 103, wherein the gNAs are gRNAs.
 105. The method ofclaim 103, wherein the gNAs are gDNAs.
 106. The method of any one ofclaims 103 to 105, wherein the the nucleic acid-guided nuclease nickaseis selected from the group consisting of CAS Class I Type I nickase, CASClass I Type III nickase, CAS Class I Type IV nickase, CAS Class II TypeII nickase, and CAS Class II Type V nickase.
 107. The method of any oneof claims 103 to 105, wherein the nucleic acid-guided nuclease nickaseis selected from the group consisting of Cas9 nickase, Cpf1 nickase,Cas3 nickase, Cas8a-c nickase, Cas10 nickase, Cse1 nickase, Csy1nickase, Csn2 nickase, Cas4 nickase, Csm2 nickase, Cmr5 nickase, Csf1nickase, C2C2 nickase, and NgAgo nickase.
 108. The method of claim 103,wherein the DNA is double stranded DNA.
 109. The method of claim 108,wherein the double stranded DNA is from genomic DNA.
 110. The method ofclaim 109, wherein the genomic DNA is human.
 111. The method of any oneof claims 103 to 110, wherein the DNA sequences are from 20 bp to 5000bp in length.
 112. The method of any one of claims 103 to 111, whereinthe targeted sites of interest are single nucleotide polymorphisms(SNPs), short tandem repeats (STRs), cancer genes, inserts, deletions,structural variations, exons, genetic mutations, or regulatory regions.113. The method of any one of claims 103 to 112, wherein the targetedsites of interest represent less than 50% of the total DNA in thesample.
 114. The method of any one of claims 103 to 113, wherein thesample is obtained from a biological sample, a clinical sample, aforensic sample, or an environmental sample.
 115. The method of any oneof claims 103 to 113, wherein the thermostable ligase capable ofcontacting the double stranded break is thermostable 5′App DNA/RNAligase or T4 RNA ligase.
 116. The method of any one of claims 103 to115, wherein the nucleic acid-guided nuclease nickase nicks the 5′ endof the DNA sequences.
 117. A method of enriching a sample for sequencesof interest, comprising: (a) providing a sample comprising sequences ofinterest and targeted sequences for depletion, wherein the sequences ofinterest comprise less than 50% of the sample; and (b) contacting thesample with a plurality of either nucleic acid-guided RNAendonuclease-gRNA complexes or a plurality of nucleic acid-guided DNAendonuclease-gDNA complexes, wherein the gRNAs and gDNAs arecomplementary to the targeted sequences, and whereby the targetedsequences are cleaved.
 118. The method of claim 117, further comprisingextracting the sequences of interest and the targeted sequences fordepletion from the sample.
 119. The method of claim 118, furthercomprising fragmenting the extracted sequences.
 120. The method of anyone of claims 117 to 119, wherein the cleaved targeted sequences areremoved by size-exclusion.
 121. The method of any one of claims 117 to120, wherein the sample is any one of a biological sample, a clinicalsample, a forensic sample or an environmental sample.
 122. The method ofany one of claims 117 to 121, wherein the sample comprises host nucleicacid sequences targeted for depletion and non-host nucleic acidsequences of interest.
 123. The method of claim 122, wherein thenon-host nucleic acid sequences comprise microbial nucleic acidsequences.
 124. The method of claim 123, wherein the microbial nucleicacid sequences are bacterial, viral or eukaryotic parasitic nucleic acidsequences.
 125. The method of any one of claims 117 to 124, wherein thegRNAs and gDNAs are complementary to ribosomal RNA sequences, splicedtranscripts, unspliced transcripts, introns, exons, or noncoding RNAs.126. The method of claim 118, wherein the extracted nucleic acidsinclude single-stranded or double-stranded RNA.
 127. The method of claim118, wherein the extracted nucleic acids include single-stranded ordouble-stranded DNA.
 128. The method of any one of claims 117 to 127,wherein the sequences of interest comprise less than 10% of theextracted nucleic acids.
 129. The method of any one of claims 117 to128, wherein the nucleic acid-guided RNA endonuclease comprises C2c2.130. The method of claim 129, wherein the C2c2 is catalytically dead.131. The method of any one of claims 117 to 128, wherein the nucleicacid-guided DNA endonuclease comprises NgAgo.
 132. The method of claim131, wherein the NgAgo is catalytically dead.
 133. The method of any oneof claims 117 to 132, wherein the sample is selected from whole blood,plasma, serum, tears, saliva, mucous, cerebrospinal fluid, teeth, bone,fingernails, feces, urine, tissue, and a biopsy.
 134. A method ofenriching a sample comprising: (a) providing a sample comprising hostnucleic acids and non-host nucleic acids; (b) contacting the sample witha plurality of nucleic acid-guided RNA endonuclease-gRNA complexes or aplurality of nucleic acid-guided DNA endonuclease-gDNA complexes,wherein the gRNAs and gDNAs are complementary to targeted sites in thehost nucleic acids, and (c) enriching the sample for non-host nucleicacids.
 135. The method of claim 134, wherein the nucleic acid-guided RNAendonuclease comprises C2c2.
 136. The method of claim 134, wherein thenucleic acid-guided RNA endonuclease comprises catalytically dead C2c2.137. The method of claim 134, wherein the nucleic acid-guided DNAendonuclease comprises NgAgo.
 138. The method of claim 134, wherein thenucleic acid-guided DNA endonuclease comprises catalytically dead NgAgo.139. The method of anyone of claims 134 to 138, wherein the host isselected from the group consisting of a human, cow, horse, sheep, pig,monkey, dog, cat, gerbil, bird, mouse, and rat.
 140. The method ofanyone of claims 134 to 138, wherein the non-host is a prokaryoticorganism.
 141. The method of anyone of claims 134 to 138, wherein thenon-host is selected from the group consisting of a eukaryote, a virus,a bacterium, a fungus, and a protozoan.
 142. The method of anyone ofclaims 134 to 141, wherein the adapter-ligated host nucleic acids andnon-host nucleic acids range from 50 bp to 1000 bp.
 143. The method ofanyone of claims 134 to 142, wherein the non-host nucleic acids compriseless than 50% of the total nucleic acids in the sample.
 144. The methodof anyone of claims 134 to 143, wherein the sample is any one of abiological sample, a clinical sample, a forensic sample or anenvironmental sample.
 145. The method of anyone of claims 134 to 144,wherein step (c) comprises reverse-transcribing the product of step (b)into cDNA.
 146. The method of anyone of claims 134 to 145, wherein step(c) comprises removing the host nucleic acids by size-exclusion. 147.The method of anyone of claims 134 to 145, wherein step (c) comprisesremoving the host nucleic acids with the use of biotin.
 148. The methodof anyone of claims 134 to 147, wherein the sample is selected fromwhole blood, plasma, serum, tears, saliva, mucous, cerebrospinal fluid,teeth, bone, fingernails, feces, urine, tissue, and a biopsy.
 149. Acomposition comprising a nucleic acid fragment, a nucleic acid-guidednuclease nickase-gNA complex, and labeled nucleotides.
 150. Thecomposition of claim 149, wherein the nucleic acid-guided nucleasenickase is selected from the group consisting of CAS Class I Type Inickase, CAS Class I Type III nickase, CAS Class I Type IV nickase, CASClass II Type II nickase, and CAS Class II Type V nickase.
 151. Thecomposition of claim 149, wherein the nucleic acid-guided nucleasenickase is selected from the group consisting of Cas9 nickase, Cpf1nickase, Cas3 nickase, Cas8a-c nickase, Cas10 nickase, Cse1 nickase,Csy1 nickase, Csn2 nickase, Cas4 nickase, Csm2 nickase, Cmr5 nickase,Csf1 nickase, C2C2 nickase, and NgAgo nickase.
 152. The composition ofany one of claims 149 to 151, wherein the gNAs are gRNAs.
 153. Thecomposition of any one of claims 149 to 151, wherein the gNAs are gDNAs.154. The composition of any one of claims 149 to 153, wherein thenucleic acid fragment comprises DNA.
 155. The composition of any one ofclaims 149 to 153, wherein the nucleic acid fragment comprises RNA. 156.The composition of any one of claims 149 to 155, wherein the nucleotidesare labeled with biotin.
 157. The composition of any one of claims 149to 156, wherein the nucleotides are part of an antibody conjugate pair.158. A composition comprising a nucleic acid fragment and acatalytically dead nucleic acid-guided nuclease-gNA complex, wherein thecatalytically dead nucleic acid-guided nuclease is fused to atransposase.
 159. The composition of claim 158, wherein thecatalytically-dead nucleic acid-guided nuclease is selected from thegroup consisting of dead CAS Class I Type I, dead CAS Class I Type III,dead CAS Class I Type IV, dead CAS Class II Type II, and dead CAS ClassII Type V.
 160. The composition of claim 158, wherein the catalyticallydead nucleic acid-guided nuclease is selected from the group consistingof dCas9, dCpf1, dCas3, dCas8a-c, dCas10, dCse1, dCsy1, dCsn2, dCas4,dCsm2, dCmr5, dCsf1, dC2C2, and dNgAgo.
 161. The composition of any oneof claims 158, 159, or 160, wherein the gNAs are gRNAs.
 162. Thecomposition of any one of claims 158 to 161, wherein the gNAs are gDNAs.163. The composition of any one of claims 158 to 162, wherein thenucleic acid fragment comprises DNA.
 164. The composition of any one ofclaims 158 to 163, wherein the nucleic acid fragment comprises RNA. 165.The composition of any one of claims 158 to 164, wherein thecatalytically dead nucleic acid-guided nuclease is fused to theN-terminus of the transposase.
 166. The composition of any one of claims158 to 165, wherein the catalytically dead nucleic acid-guided nucleaseis fused to the C-terminus of the transposase.
 167. A compositioncomprising a nucleic acid fragment comprising methylated nucleotides, anucleic acid-guided nuclease nickase-gNA complex, and unmethylatednucleotides.
 168. The composition of claim 167, wherein the nucleicacid-guided nuclease nickase is selected from the group consisting ofCAS Class I Type I nickase, CAS Class I Type III nickase, CAS Class IType IV nickase, CAS Class II Type II nickase, and CAS Class II Type Vnickase.
 169. The composition of claim 167, wherein the nucleicacid-guided nuclease nickase is selected from the group consisting ofCas9 nickase, Cpf1 nickase, Cas3 nickase, Cas8a-c nickase, Cas10nickase, Cse1 nickase, Csy1 nickase, Csn2 nickase, Cas4 nickase, Csm2nickase, Cmr5 nickase, Csf1 nickase, C2C2 nickase, and NgAgo nickase.170. The composition of any one of claims 167, 168, or 169, wherein thegNAs are gRNAs.
 171. The composition of any one of claims 167 to 170,wherein the gNAs are gDNAs.
 172. The composition of any one of claims167 to 171, wherein the nucleic acid fragment comprises DNA.
 173. Thecomposition of any one of claims 167 to 171, wherein the nucleic acidfragment comprises RNA.
 174. The composition of any one of claims 167 to173, wherein the nucleotides are labeled with biotin.
 175. Thecomposition of any one of claims 167 to 174, wherein the nucleotides arepart of an antibody conjugate pair.